Multifunctional oral vaccine based on chromosome recombineering

ABSTRACT

A recombineered  Salmonella typhi  Ty21a, compositions and vaccines comprising such a Ty21a, and a method for recombineering comprising inserting a large antigenic region into a bacterial chromosome for the purpose of making multivalent vaccines to protect against one or more disease agents are described herein.

RELATED APPLICATION DATA

This application is a 35 U.S.C. §371 national phase application ofPCT/US2013/059980, filed on Sep. 16, 2013, entitled “MULTIFUNCTIONALORAL VACCINE BASED ON CHROMOSOME RECOMBINEERING”, which applicationclaims the priority benefit of U.S. Provisional Application No.61/701,939, filed Sep. 17, 2012, entitled “MULTIFUNCTIONAL ORAL VACCINEBASED ON CHROMOSOME RECOMBINEERING”, all of which are incorporatedherein by reference in their entirety. Each of the applications andpatents cited in this text, as well as each document or reference citedin each of the applications and patents (including during theprosecution of each issued patent; “application cited documents”), andeach of the PCT and foreign applications or patents corresponding toand/or claiming priority from any of these applications and patents, andeach of the documents cited or referenced in each of the applicationcited documents, are hereby expressly incorporated herein by referenceand may be employed in the practice of the invention. More generally,documents or references are cited in this text, either in a ReferenceList before the claims, or in the text itself; and, each of thesedocuments or references (“herein cited references”), as well as eachdocument or reference cited in each of the herein cited references(including any manufacturer's specifications, instructions, etc.), ishereby expressly incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

The instant application was made with government support; the governmenthas certain rights in this invention.

SEQUENCE LISTING DATA

The Sequence Listing text file attached hereto, created Sep. 12, 2013,size 144 kilobytes, filename “6137FDA9PCT_SEQ_Listing_20130912_ST25.txt”is incorporated herein by reference in its entirety.

BACKGROUND

Bacillary dysentery and enteric fevers continue to be important causesof morbidity in both developed and developing nations. Shigella cause anestimated >150 million cases of dysentery, and enteric fever occursin >27 million people, annually (Bardhan, et al. (2010) Emerging InfecDiseases 16:1718-1723; Crump, et al. (2004) Bulletin of the WHO82:346-353; Crump and Mintz (2010) Clin Infec Diseases 50:241-246; WHO(2005) Guidelines for the control of shigellosis . . . ). Shigellosisand enteric fevers together cause >250,000 deaths annually (Bardhan, etal. (2010) Emerging Infec Diseases 16:1718-1723; Crump, et al. (2004)Bulletin of the WHO 82:346-353), demonstrating a continuing need for amulti-valent vaccine for protection against these diseases. Importantly,two thirds of all Shigella cases occur in children under the age of fiveyears (Kotloff et al. (1999) Bull. WHO, 77:651-666; Kweon (2008) Curr.Opin. Infect. Dis., 21:313-318), and enteric fevers are most common inyoung school-age children (Crump and Mintz (2010) Clin Infec Diseases50:241-246).

The genus Shigella includes four species; S. dysenteriae, S. flexneri,S. boydii and S. sonnei, also designated as serogroups A, B, C and D,respectively. The first three species, respectively, are further dividedinto serotypes based upon differences in LPS structures. Upon ingestionof contaminated food or water, Shigella cause an acute invasiveinfection of the large intestine that typically results in severeabdominal cramps, fever, and dysentery (i.e., small volume <5 ml stoolscomprised of mucus, polymorphonuclear neutrophils, necrotic tissue, andstreaks of blood). S. boydii and S. sonnei oftentimes cause a milderdisease when compared to S. dysenteriae and S. flexneri. S. flexneri isresponsible for most endemic infections in developing countries. S.sonnei is the species responsible for most endemic infections observedin industrialized countries. The US CDC estimates an incidence of˜450,000 cases of S. sonnei disease in the US each year, which occursmostly in child daycare facilities. S. sonnei is also responsible for aconsiderable amount of morbidity in developing countries such asThailand, where it is the cause of ˜95% of shigellosis (Mead et al.(1999) Emerg. Infect. Dis., 5:607-625; Putthasri et al. (2009) Emerg.Infect. Dis., 15:423-432). S. dysenteriae serotype 1 (Sd1) is especiallyimportant, as it causes severe dysentery plus hemolytic uremic syndrome(as a result of producing the potent cytotoxin Shiga toxin), typicallyresulting in a higher mortality rate than infections due to otherShigella species. Furthermore, Sd1 classically causes large epidemicswith high attack rates (World Health Organization (2005) Guidelines forthe Control of Shigellosis, Including Epidemics Due to Shigelladysenteriae Type 1, ISBN 924159330X).

Currently, there is no licensed vaccine available to prevent theoccurrence of shigellosis. Increasing multiple antibiotic resistance inShigella commonly thwarts local therapies. As a result, the World HealthOrganization considers development of a vaccine against shigellosis atop priority. Most importantly, there is a global public health need fora vaccine to prevent shigellosis in endemic populations, travelers, andthe military. Due to the existence of a large number of Shigellaserotypes (>40), some investigators have attempted to find surfaceantigens common to most serotypes (Kaminski et al. (2009) Expert Rev.Vaccines, 8:1693-1704). Despite significant efforts, common surfaceproteins by themselves (e.g., ipaA,B,C,D) do not appear to stimulatesignificant or sustained protective immunity to Shigella infection.However, there is considerable evidence that protective immunity isdirected primarily against Shigella serotype specific LPS O-antigen,which highlights the importance of O-antigens as targets for vaccinedevelopment (Ferreccio et al. (1991) Am. J. Epidemiol., 134:614-627;DuPont et al. (1972) J. Infect. Dis., 125:5-11; DuPont et al. (1972) J.Infect. Dis., 125:12-16).

Indeed, lipopolysaccharide (LPS) alone has been shown to be a potentvaccine antigen for specific protection against shigellosis. The plasmidcloning of heterologous LPS biosynthetic genes and the expression inTy21a of either S. sonnei or of S. dysenteriae 1 LPS's have previouslybeen reported. The resulting plasmids encoding Shigella LPSs werereasonably stable for more than 50 generations of growth innon-selective media, but they still contained an objectionableantibiotic resistance marker. The deletion of this antibiotic resistancemarker resulted in significant plasmid instability.

Based upon specific Shigella serotype prevalence worldwide and previousstudies of serotype cross-protection among Shigellae, Noriega, et al.(1999 Infection and Immunity 67:782-788) have suggested that amultivalent vaccine containing LPSs of S. Sonnei, S. dysenteriae 1, S.flexneri 2a, S. flexneri 3a, and S. flexneri 6 could protect against˜85%of shigellosis worldwide.

The live, attenuated, oral vaccine Salmonella enterica serovar Typhistrain Ty21a has been utilized extensively as a broad-based oral vaccinevector for the expression of various foreign antigens (Xu et al. (2007)Vaccine, 25:6167-6175; Xu et al. (2002) Infect. Immun., 70:4414-4423;Osorio et al. (2009) Infect. Immun., 44:1475-1482). Ty21a is the onlylicensed, live, attenuated vaccine for protection against typhoid fever.Moreover, it has been safely administered to more than 200 millionrecipients around the world. As a whole-cell vaccine, Ty21a inducesmucosal, humoral, and cellular immunity, leading to high-level,long-term protection (i.e., virtually undiminished protection at the endof 7 full years) against typhoid fever (Levine, et al. (1999) Vaccine 17Suppl 2: S22-27) with considerable evidence of cross-protection againstboth S. Paratyphi A and B (Bardhan, et al. (2010) Emerging InfecDiseases 16:1718-1723; D'Amelio et al. (1988) Infect. Immun.,56:2731-2735; Levine, et al. (2007) Clin Infec Diseases 45 Supp1:S24-28; Schwartz, et al. (1990) Archives Internal Med 150:349-351;Wahid, et al. (2012) Clin and Vaccine Immunol CVI 19:825-834).

SUMMARY

In earlier vaccine efforts, the 180 kb form 1 O-antigen encoding plasmidof S. sonnei was transferred, as a proof-of-principle, as part of alarge˜300 kb plasmid cointegrate to the vaccine strain Ty21a. Theresulting hybrid vaccine strain, 5076-1C, expressed both homologous S.typhi 9,12 O-antigen and heterologous S. sonnei O-antigen that wereimmunogenic (Seid et al. (1984) J. Biol. Chem., 259:9028-9034; Formal etal. (1981) Infect. Immun., 34:746-750). Importantly, oral immunizationof volunteers with 5076-1C was safe and elicited significant protectionagainst virulent S. sonnei oral challenge (i.e., 100% protection againstsevere dysentery) (Black et al. (1987) J. Infect. Dis., 155:1260-1265;Herrington et al. (1990) Vaccine, 8:353-357; Van de Verg et al. (1990)Infect. Immun., 58:2002-2004). Unfortunately, though not entirelyunexpectedly, the considerable protection afforded volunteers receivingthe first two lots of vaccine was not observed with subsequent vaccinelots, due to loss of the form I gene region from the geneticallyunstable, large cointegrate plasmid in 5076-1C (Formal et al. (1981)Infect. Immun., 34:746-750). Thus, further molecular studies werecarried out to stabilize the S. sonnei form I gene region in vaccinevector constructs. In these subsequent studies, the minimal size generegions needed for stable expression of S. sonnei or S. dysenteriaeserotype 1 O-antigens were determined to be between 11 and 15 kb (Xu etal. (2007) Vaccine, 25:6167-6175; Xu et al. (2002) Infect. Immun.,70:4414-4423). These studies also revealed that the S. sonnei O-antigencould be expressed as core-linked LPS or as a Group 4 O-antigen capsulein either Ty21a or in Shigella, as are LPSs of certain other Shigellaserotypes and of Salmonella Typhi, but that either form of O-antigenexpression appears to be equally immunogenic.

These early Shigella LPS constructs were made in the low copygenetically stable plasmid pGB-2, which carries an antibiotic resistancemarker for genetic utility. Removal of this antibiotic resistance markerto facilitate human vaccine use proved problematic, and resulted inunexplained lower plasmid genetic stability (i.e., the antibioticresistant plasmid was stable during growth in the absence of antibioticpressure for more than 60 generations, but removal of just theantibiotic resistance gene sequences somehow resulted in geneticinstability).

Thus, a study was undertaken to address the issues of genetic stabilityand the need for a selectable, but removable antibiotic resistancemarker. Towards this end, the previously described recombinationtechniques (Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA,97:6640-6645 were modified to recombineer a >11 kb Shigella sonneiO-antigen biosynthetic gene region into the Salmonella Typhi Ty21achromosome to construct Ty21a-Ss (Ty21a expressing S. sonnei O-antigen).Further, this chromosomal integration of essential S. sonnei LPSbiosynthetic genes was 100% genetically stable, even after removal of aselectable antibiotic resistance gene cassette. Heterologous S. sonneiform 1 LPS was stably expressed in Ty21a-Ss along with homologousSalmonella Typhi O-antigen. Of note, the candidate vaccine strainTy21a-Ss elicited solid immune protection in mice against virulent S.sonnei challenge.

In one aspect, the invention provides a Salmonella typhi Ty21acomprising a Shigella sonnei O-antigen biosynthetic gene region insertedinto the Salmonella typhi Ty21a chromosome, wherein: a) heterologousShigella sonnei form 1 O-antigen is stably expressed together with orwithout homologous Salmonella typhi O-antigen; b) immune protection iselicited against virulent Shigella sonnei challenge; and c) immuneprotection is elicited against virulent Salmonella Typhi challenge. Inanother aspect, the invention provides a Salmonella typhi Ty21acomprising a Shigella sonnei O-antigen biosynthetic gene region insertedinto the Salmonella typhi Ty21a chromosome, wherein: a) heterologousShigella sonnei form 1 O-antigen is stably expressed together withhomologous Salmonella typhi O-antigen; b) immune protection is elicitedagainst virulent Shigella sonnei challenge; and c) immune protection iselicited against virulent Salmonella Typhi challenge.

In one embodiment of the invention, the region is encoded by a DNAsequence selected from the group consisting of: a) a DNA sequence as setout in SEQ ID NO:2; b) a DNA sequence that shares at least about 90%sequence identity with the DNA sequence set out in SEQ ID NO:2; and c) aDNA sequence that is a functional variant of the DNA sequence set out inSEQ ID NO:2. SEQ ID NO:2 is provided herein and has been submitted toGENBANK and designated JX436479 but will not be released untilpublication of a manuscript describing portions of this study. Thesequence with accession number AF294823 (SEQ ID NO:23) actually containsabout 19 ORFs and includes the IS elements. In the current study, onlythe essential 10 ORFs (out of the 19 ORFs displayed in AF294823 (SEQ IDNO:23)) were cloned and inserted into the Ty21a chromosome.

In a further embodiment of the invention, the Ty21a further comprises anO-antigen biosynthetic gene region from a bacterial strain selected fromthe group consisting of: Shigella species (Shigella dysenteriae,Shigella flexneri, and Shigella boydii), Escherichia coli serotypes,Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacterspecies, Yersinia species, and Pseudomonas species.

In another aspect, the invention provides a Salmonella typhi Ty21acomprising a Shigella dysenteriae 1 O-antigen biosynthetic gene regioninserted into the Salmonella typhi Ty21a chromosome, wherein: a)heterologous Shigella dysenteriae serotype 1 O-antigen is stablyexpressed together with or without homologous Salmonella typhiO-antigen; b) immune protection is elicited against virulent Shigelladysenteriae 1 challenge; and c) immune protection is elicited againstvirulent Salmonella Typhi challenge. In still another aspect, theinvention provides a Salmonella typhi Ty21a comprising a Shigelladysenteriae 1 O-antigen biosynthetic gene region inserted into theSalmonella typhi Ty21a chromosome, wherein: a) heterologous Shigelladysenteriae serotype 1 O-antigen is stably expressed together withhomologous Salmonella typhi O-antigen; b) immune protection is elicitedagainst virulent Shigella dysenteriae 1 challenge; and c) immuneprotection is elicited against virulent Salmonella Typhi challenge.

In one embodiment of the invention, the region is encoded by a DNAsequence selected from the group consisting of: a) a DNA sequence as setout in SEQ ID NOs:3 or 4; b) a DNA sequence that shares at least about90% sequence identity with the DNA sequence set out in SEQ ID NO:3 or 4;and c) a DNA sequence that is a functional variant of the DNA sequenceset out in SEQ ID NO:3 or 4.

In a preferred embodiment of the invention, the region is encoded by aDNA sequence selected from the group consisting of: a) a DNA sequence asset out in SEQ ID NO:33; b) a DNA sequence that shares at least about90% sequence identity with the DNA sequence set out in SEQ ID NO:33; andc) a DNA sequence that is a functional variant of the DNA sequence setout in SEQ ID NO:33.

In still another embodiment of the invention, the Ty21a furthercomprises an O-antigen biosynthetic gene region from a bacterial strainselected from the group consisting of: Shigella species (Shigellasonnei, Shigella flexneri, and Shigella boydii), Escherichia coliserotypes, Salmonella enterica serovars, Vibrio cholerae serotypes,Enterobacter species, Yersinia species, and Pseudomonas species.

In another aspect, the invention provides a plasmid construct having i)a DNA sequence as set out in SEQ ID NO: 1 or ii) a DNA sequence thatshares at least about 90% sequence identity with the DNA sequence setout in SEQ ID NO:1. In a further embodiment of the invention, theplasmid construct further comprises a Shigella sonnei O-antigenbiosynthetic gene region. In still a further embodiment of theinvention, the plasmid construct further comprises a Shigelladysenteriae 1 O-antigen biosynthetic gene region.

In another aspect, the invention provides a method of recombineering alarge antigenic gene region into a bacterial chromosome, comprising: i)cloning the region into a vector containing: ia) a geneticallyselectable marker flanked 5′ and 3′ by an FRT site, respectively; ib) amultiple cloning site downstream of the 3′ FRT site; and ic) two sitesof chromosome homology, one of the two located upstream of the 5′ FRTsite, and one of the two located downstream of the multiple cloningsite; ii) integrating the region into the bacterial chromosome using λred recombination; iii) selecting for the genetically selectable marker;and iv) removing the selectable marker (e.g., gene), thus recombineeringthe region into the chromosome. In one embodiment of the inventivemethod, the genetically selectable marker is an antibiotic resistancemarker. In another embodiment of the inventive method, the selectablemarker is rendered non-functional (rather than removed). In anotherembodiment of the inventive method, the bacterial chromosome can bevaried. In still another embodiment of the inventive method, theinsertion site (within the chromosome) can be varied.

In yet another embodiment of the inventive method, the antigenic generegion is about 5 to about 20 kb long. In still another embodiment ofthe inventive method, the antigenic region is longer than 20 kb. Inanother embodiment of the inventive method, the vector is selected fromthe group consisting of a plasmid, phage, phasmid, and cosmid construct.In another embodiment, the plasmid construct is the above-disclosedplasmid construct having i) a DNA sequence as set out in SEQ ID NO: 1 orii) a DNA sequence that shares at least about 90% sequence identity withthe DNA sequence set out in SEQ ID NO:1.

In another embodiment of the inventive method, the bacterial chromosomeis Salmonella typhi Ty21a. In still another embodiment of the inventivemethod, the antibiotic resistance marker is kanamycin. In still anotherembodiment of a method according to the invention, the antigenic regionis a Shigella sonnei O-antigen biosynthetic gene region. In yet anotherembodiment of a method according to the invention, the antigenic regionis a Shigella dysenteriae 1 O-antigen biosynthetic gene region. In yetanother embodiment of a method according to the invention, the antigenicregion is a Shigella flexneri 2a O-antigen biosynthetic gene region. Inyet another embodiment of a method according to the invention, theantigenic region is a Shigella flexneri 3a O-antigen biosynthetic generegion.

In another embodiment, the inventive method is for use in a whole cellbacterial vaccine. In still another embodiment, the inventive method isfor use in a live attenuated Salmonella strain, a Shigella strain, aListeria strain, a Yersinia strain, an Escherichia coli strain, anEnterobacteriaceae strain, in a protozoan strain, or in another livevectored vaccine.

In still another embodiment of a method according to the invention, theregion is engineered between about 500 to about 1000 bp regions ofbacterial chromosome homology before step ii. In still anotherembodiment of a method according to the invention, the kanamycinresistance gene is removed via recombination induced followingtransformation with pCP20.

In one aspect, the invention provides a composition of matter comprisingthe herein-disclosed Salmonella typhi Ty21a in combination with aphysiologically acceptable carrier.

In another aspect, the invention provides a vaccine comprising theherein-disclosed Salmonella typhi Ty21a in combination with aphysiologically acceptable carrier.

In yet another aspect, the invention provides a method of preventing ortreating at least one bacterial infection comprising administering aprophylactically or therapeutically effective amount of theherein-disclosed Salmonella typhi Ty21a to a subject, thus preventing ortreating the at least one bacterial infection.

In another embodiment of the inventive Salmonella typhi Ty21a, the generegion is partially or wholly chemically synthesized.

In another embodiment of the inventive plasmid construct, the DNAsequence is partially or wholly chemically synthesized.

In one aspect, the invention provides the use of an O-antigenbiosynthetic gene region in a bacterial strain designed to biologicallymanufacture protein-LPS conjugate products.

In an additional embodiment of the invention, the gene clusterresponsible for the biosynthesis of the bacterial LPS, capsulepolysaccharide (CPS), or oligo polysaccharides (oligo PS) is transformedinto E. coli together with the protein carrier of interest along with anenzyme that performs the bioconjugation reaction in vivo. Once producedupon induction, simple purification steps are performed, and thebiological products are formulated for use as a vaccine (Dro (2012) Gen.Eng. Biotech. News, Vol. 32,www.genengnews.com/gen-articles/developing-next-gen-conjugate-vaccines/4042;Gambillara (2012) BioPharm Int., 25:28-32; Kowarik et al. (2006)Science, 314:1148-1150; Wacker et al. (2006) Proc. Natl. Acad. Sci. USA,103:7088-7093; Kowarik et al. (2006) EMBO J., 25:1957-1966; Feldman etal. (2005) Proc. Natl. Acad. Sci. USA, 102:3016-3021; Nita-Lazar et al.(2005) Glycobiology, 15:361-367; Wacker et al. (2002) Science,298:1790-1793; Wacker (2002) N-linked Protein Glycosylation: FromEukaryotes to Bacteria, Ph.D. thesis submitted to the Swiss FederalInstitute of Technology, Zurich).

The methods according to the invention will, in additional embodiments,allow for the insertion of antigenic gene region(s) into other entericbacteria, as well.

The Salmonella serovars Typhi and Paratyphi are acquired throughingestion of contaminated food and water and cause indistinguishableenteric fevers. Though peak incidence occurs in young school children,enteric fever affects all ages. Multiple antibiotic resistancecomplicates treatment and makes vaccination a desired priority in areaslacking proper sanitation. Although two typhoid vaccines are available,there has been little effort to introduce them on a large scale in thedeveloping world. A multivalent vaccine that would simultaneouslyprotect against enteric fevers and shigellosis would have greatpractical advantages in advancing immunization against these diseases,not only to travelers and the military, but also to the developingnations which suffer high resulting disease mortality.

In yet another aspect, the invention provides a Salmonella typhi Ty21acomprising a Shigella flexneri 2a O-antigen biosynthetic gene regioninserted into the Salmonella typhi Ty21a chromosome, wherein: a)heterologous Shigella flexneri 2a O-antigen is stably expressed togetherwith or without homologous Salmonella typhi O-antigen; b) immuneprotection is elicited against virulent Shigella flexneri 2a challenge;and c) immune protection is elicited against virulent Salmonella typhichallenge. In still another aspect, the invention provides a Salmonellatyphi Ty21a comprising a Shigella flexneri 2a O-antigen biosyntheticgene region inserted into the Salmonella typhi Ty21a chromosome,wherein: a) heterologous Shigella flexneri 2a O-antigen is stablyexpressed together with homologous Salmonella typhi O-antigen; b) immuneprotection is elicited against virulent Shigella flexneri 2a challenge;and c) immune protection is elicited against virulent Salmonella typhichallenge.

In still another aspect, the invention provides a Salmonella typhi Ty21acomprising a Shigella flexneri 3a O-antigen biosynthetic gene regioninserted into the Salmonella typhi Ty21a chromosome, wherein: a)heterologous Shigella flexneri 3a O-antigen is stably expressed togetherwith or without homologous Salmonella typhi O-antigen; b) immuneprotection is elicited against virulent Shigella flexneri 3a challenge;and c) immune protection is elicited against virulent Salmonella typhichallenge. In yet another aspect, the invention provides a Salmonellatyphi Ty21a comprising a Shigella flexneri 3a O-antigen biosyntheticgene region inserted into the Salmonella typhi Ty21a chromosome,wherein: a) heterologous Shigella flexneri 3a O-antigen is stablyexpressed together with homologous Salmonella typhi O-antigen; b) immuneprotection is elicited against virulent Shigella flexneri 3a challenge;and c) immune protection is elicited against virulent Salmonella typhichallenge.

Other aspects of the invention are described in or are obvious from thefollowing disclosure and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of Examples, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, in which:

FIG. 1 provides a schematic representation of the cloning andchromosomal integration of S. sonnei form I LPS biosynthetic genes.Panel A shows the ORFs involved in form 1 O-antigen biosynthesis andadjacent IS elements from the cloned insert of pXK65, reportedpreviously (Xu, et al. (2002) Infect and immunity 70:4414-4423). InPanel B, the latter part of wzz (orf3), containing the promoter of theO-antigen operon, through wzy(orf7) was PCR-amplified as fragment A andcloned into HindIII and BamHI sites of pMD-TV (SEQ ID NO:1). Next, thegenes wbgV (orf9) through apqZ (orf14) were PCR-amplified as fragment Band cloned into the BamHI and XhoI sites adjacent to fragment A. InPanel C, the resulting plasmid pMD-TV-Ss-1, was used as a template toPCR-amplify tviD through vexA using forward (F) and reverse (R) primers.In Panel D, the PCR product was integrated into the tviD-tviE-vexAregion of the Ty21a chromosome by λ red-mediated recombination. Finally,in Panel E, the Kan^(r) cassette was removed following temporaryintroduction of the temperature-sensitive plasmid pCP20 by expression ofFLP recombinase, generating the final antibiotic-sensitive, chromosomalintegrant Ty21a-Ss (strain MD77).

FIG. 2 shows the results of analyses of SDS-PAGE-separatedpolysaccharide isolated from various Shigella sonnei, E. coli, and Ty21aisolates by, in Panel A, silver staining and in Panel B, Westernimmunoblotting with form 1-specific antibody.

FIG. 3 shows bar graphs depicting mouse serum IgG responses, measured byELISA, to S. sonnei LPS (FIG. 3A) and Ty21a LPS (FIG. 3B) afterintraperitoneal immunization of mice with PBS, Ty21a, or vaccine strainTy21a-Ss simultaneously expressing both Ty21a LPS and S. sonnei LPS.ELISA plates were coated with S. sonnei LPS or Ty21a LPS and blockedwith 1% BSA. Serial dilutions of serum collected from vaccinated micewere added to the microtiter wells, washed, and LPS-bound IgG antibodieswere detected using anti-mouse IgG-HPR. End point titers for the threegroups of mice that received PBS, Ty21a, or Ty21a-Ss are shown following1, 2, or 3 vaccine doses. Each symbol represents a single mouse, andbars represent the mean±SEM of each group of 10 mice.

FIG. 4 shows a schematic diagram depicting S. dysenteriae biosyntheticgenes integrated into Vi capsule operon of Ty21a vaccine strain.

FIG. 5 provides a schematic representation of the cloning andchromosomal integration of S. dysenteriae O-antigen genes. The figureshows how the lpp promoter was inserted to replace the native promoter.

FIG. 6 shows S. dysenteriae O-antigen expression as determined viaWestern blotting.

FIG. 7 shows mouse immunogenicity determined by ELISA. FIG. 7Aquantifies the IgG elicited against S. dysenteriae LPS. FIG. 7Bquantifies the IgG elicited against S. Typhi 9,12 LPS.

FIG. 8 shows a plot of data for immunized mice challenged IP with S.dysenteriae 1 at a dose of approximately 10×LD₅₀.

FIG. 9 shows the chemical composition of the different serotypes ofShigella flexneri (modified from Allison, et al. 2000 Trends inMicrobiol 8:17-23).

FIG. 10 shows Silver stain (FIG. 10A) and Western blot (FIGS. 10B and10C) with anti-2a (FIG. 10B) and anti-3a (FIG. 10C) antibodies.

FIG. 11 is a schematic representation of S. flexneri basic O-antigenbackbone, serotype Y, and the genes involved in the biosynthesis of thisO-antigen backbone are located in the rfb operon (˜10 kb) were clonedinto pMD-TV plasmid.

FIG. 12 shows a Western blot with anti-S. flexneri 3a antibody.

FIG. 13 shows a Western blot with anti-S. flexneri 2a antibody.

FIG. 14 provides schematic representations of Ty21a-Y (MD114), Ty21a-2a(MD194), Ty21a-2al (MD212), and Ty21a-3a (MD196).

DETAILED DESCRIPTION

In an effort to elucidate a method to insert the large >10 kb S. sonneiLPS gene region into the chromosome that would allow for removal of aselectable marker and would result in 100% genetic stability, anexisting recombination method was optimized to mediate the insertion ofa >11 kb region encoding the S. sonnei LPS genes into the Ty21a genomein a region that does affect normal cell metabolism. This chromosomalinsert was shown to be 100% genetically stable. Further testingdemonstrated that the resulting strain Ty21a-Ss simultaneously expressesboth homologous Ty21a and heterologous S. sonnei O-antigens. Moreover,Ty21a-Ss elicited a strong dual anti-LPS serum immune response and 100%protection in mice against a virulent S. sonnei challenge. This newvaccine candidate, absolutely stable for vaccine manufacture, shouldprovide combined protection against enteric fevers due to Salmonellaserovar Typhi (and some Paratyphi infections) and against shigellosisdue to S. sonnei.

Definitions

The terms “O-Ps (O polysaccharide) biosynthesis” and “O-antigenbiosynthetic” genes are used interchangeably herein. The O-antigen is apolysaccharide that can be expressed as a core-linked lipopolysaccharide(LPS) or as a group 4 capsule where the O-antigen is expressed at thecell surface but linked to a lipid other than lipid A-coreoligosaccharide.

The terms “vector” and “vehicle” are used interchangeably in referenceto nucleic acid molecules that transfer DNA segment(s) from one cell toanother. The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression (i.e.,transcription and/or translation) of an operably linked coding sequencein a particular host organism. Expression vectors are exemplified by,but not limited to, plasmids, plasmid constructs, plasmids, phagemids,shuttle vectors, cosmids, viral vectors, the chromosome, mitochondrialDNA, plastid DNA, and nucleic acid fragments, that may be used forexpression of a desired sequence in a cell, such as a human cell, aviancell, a fungal cell, a protozoan cell, and/or insect cell. Nucleic acidsequences used for expression in prokaryotes include a promoter,optionally an operator sequence, a ribosome-binding site and possiblyother sequences. Eukaryotic cells are known to utilize promoters,enhancers, and termination and polyadenylation signals. FlippaseRecognition Target (FRT) sites and multiple cloning sites (MCS), alsocalled polylinkers, are also utilized. A MCS is a short DNA segmentcontaining many (up to about 20) restriction sites that are typicallyunique, occurring only once within a given plasmid.

The term “transformation” as used herein, refers to any mechanism bywhich a DNA molecule (i.e., plasmid or PCR amplicon) may be incorporatedinto a host cell. A successful transformation results in the capabilityof the host cell to express any operative genes carried by the plasmidor incorporated into the chromosome. Transformations may result ingenetically stable or transient gene expression. One example of atransient transformation comprises a plasmid vector within a cell,wherein the plasmid is not integrated into the host cell chromosome andis segregated from cells during cell division. Alternatively, a stabletransformation comprises a plasmid within a cell, wherein the plasmid isintegrated within the host cell genome or exists stably in the cytoplasmafter many cell divisions.

The terms “subject”, “patient”, and “individual”, as used herein,interchangeably refer to a multicellular animal (including mammals(e.g., humans, non-Human primates, murines, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.),avians (e.g., chicken), amphibians (e.g. Xenopus), reptiles, and insects(e.g. Drosophila). “Animal” includes guinea pig, hamster, ferret,chinchilla, mouse and cotton rat.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST®, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul et al.(1990) J. Mol. Biol., 215:403-410; Altschul, et al., Methods inEnzymology; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402;Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds.),Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press, 1999; all of which are incorporated herein byreference. In addition to identifying homologous sequences, the programsmentioned above typically provide an indication of the degree ofhomology.

The phrases “homology” or “substantial homology”, as used herein, refersto a comparison between amino acid sequences. As will be appreciated bythose of ordinary skill in the art, two sequences are generallyconsidered to be “substantially homologous” if they contain homologousresidues in corresponding positions. Homologous residues may beidentical residues. Alternatively, homologous residues may benon-identical residues with appropriately similar structural and/orfunctional characteristics. For example, as is well known by those ofordinary skill in the art, certain amino acids are typically classifiedas “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar”or “non-polar” side chains. Substitution of one amino acid for anotherof the same type may often be considered a “homologous” substitution.

In some embodiments, two sequences are considered to be substantiallyhomologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their correspondingresidues are homologous over a relevant stretch of residues. In someembodiments, the relevant stretch is a complete sequence. In someembodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or moreresidues.

The phrases “identity” or “substantial identity”, as used herein, referto a comparison between amino acid or nucleic acid sequences. As will beappreciated by those of ordinary skill in the art, two sequences aregenerally considered to be “substantially identical” if they containidentical residues in corresponding positions. Indeed, “identity” refersto the overall relatedness between polymeric molecules, e.g., betweennucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/orbetween polypeptide molecules. Calculation of the percent identity oftwo nucleic acid sequences can, for example, be performed by aligningthe two sequences for optimal comparison purposes (e.g., gaps can beintroduced in stretches of one or both of a first and a second nucleicacid sequence for optimal alignment and non-identical sequences can bedisregarded for comparison purposes). In certain embodiments, the lengthof a sequence aligned for comparison purposes is at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or substantially 100% of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which needs to be introduced foroptimal alignment of the two sequences. The comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm.

In some embodiments, two sequences are considered to be substantiallyidentical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their correspondingresidues are identical over a relevant stretch of residues. In someembodiments, the relevant stretch is a complete sequence. In someembodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or moreresidues.

Reference herein to any numerical range (for example, a dosage range)expressly includes each numerical value (including fractional numbersand whole numbers) encompassed by that range. For example, but withoutlimitation, reference herein to a range of “2×10⁹ to 5×10¹⁰” CFU(colony-forming units) includes all whole numbers of and fractionalnumbers between the two. In a further illustration, reference herein toa range of “less than x” (wherein x is a specific number) includes wholenumbers x−1, x−2, x−3, x−4, x−5, x−6, etc., and fractional numbersx−0.1, x−0.2, x−0.3, x−0.4, x−0.5, x−0.6, etc. In yet anotherillustration, reference herein to a range of from “x to y” (wherein x isa specific number, and y is a specific number) includes each wholenumber of x, x+1, x+2 . . . to y−2, y−1, y, as well as each fractionalnumber, such as x+0.1, x+0.2, x+0.3 . . . to y−0.2, y−0.1. In anotherexample, the term “at least 95%” includes each numerical value(including fractional numbers and whole numbers) from 95% to 100%,including, for example, 95%, 96%, 97%, 98%, 99% and 100%.

The terms “antigen,” “immunogen,” “antigenic,” “immunogenic,”“antigenically active,” “immunologic,” and “immunologically active”, asused herein, refer to any substance that is capable of inducing aspecific immune response (including eliciting a soluble antibodyresponse) and/or cell-mediated immune response (including eliciting acytotoxic T-lymphocyte (CTL) response).

An individual referred to as “suffering from” a disease, disorder,and/or condition (e.g., influenza or typhoid fever infection) herein hasbeen diagnosed with and/or displays one or more symptoms of a disease,disorder, and/or condition.

As used herein, the term “at risk” for disease (such as bacterialinfection), refers to a subject (e.g., a human) that is predisposed tocontracting the disease and/or expressing one or more symptoms of thedisease. Such subjects include those at risk for failing to elicit animmunogenic response to a vaccine against the disease. Thispredisposition may be genetic (e.g., a particular genetic tendency toexpressing one or more symptoms of the disease, such as heritabledisorders, the presence of bacterial species blocking antibodies, thepresence of reduced levels of bactericidal antibodies, etc.), or due toother factors (e.g., immune suppressive conditions, environmentalconditions, exposures to detrimental compounds, including immunogens,present in the environment, etc.). The term subject “at risk” includessubjects “suffering from disease,” i.e., a subject that is experiencingthe disease. It is not intended that the present invention be limited toany particular signs or symptoms. Thus, it is intended that the presentinvention encompasses subjects that are experiencing any range ofdisease, from sub-clinical infection to full-blown disease, wherein thesubject exhibits at least one of the indicia (e.g., signs and symptoms)associated with the disease.

The terms “treat,” “treatment,” or “treating”, as used herein, refer toany method used to partially or completely alleviate, ameliorate,relieve, inhibit, prevent, delay onset of, reduce severity of and/orreduce incidence of one or more symptoms or features of a disease,disorder, and/or condition (e.g., bacterial infection). Treatment may beadministered to a subject who does not exhibit signs of a disease,disorder, and/or condition. In some embodiments, treatment may beadministered to a subject who exhibits only early signs of the disease,disorder, and/or condition for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

As used herein, the terms “immunogenically effective amount,”“immunologically effective amount”, and “antigenically effective amount”refer to that amount of a molecule that elicits and/or increasesproduction of an immune response (including production of specificantibodies and/or induction of a TCL response) in a host uponvaccination. It is preferred, though not required, that theimmunologically-effective (i.e., immunogenically effective) amount is a“protective” amount. The terms “protective” and “therapeutic” amount ofa composition or vaccine refer to an amount of the composition orvaccine that prevents, delays, reduces, palliates, ameliorates,stabilizes, and/or reverses disease (for example, bacterial infection)and/or one or more symptoms of disease.

As used herein, the term “vaccination” refers to the administration of acomposition or vaccine intended to generate an immune response, forexample, to a disease-causing agent. For the purposes of the presentinvention, vaccination can be administered before, during, and/or afterexposure to a disease-causing agent, and, in certain embodiments,before, during, and/or shortly after exposure to the agent. In someembodiments, vaccination includes multiple administrations,appropriately spaced in time, of a vaccinating composition (vaccine).

The terms “comprises”, “comprising”, are intended to have the broadmeaning ascribed to them in US Patent Law and can mean “includes”,“including” and the like.

The invention can be understood more fully by reference to the followingdetailed description and illustrative examples, which are intended toexemplify non-limiting embodiments of the invention.

Additional Embodiments of the Invention

In one embodiment, the invention comprises a prokaryotic microorganism.In another embodiment, the prokaryotic microorganism is a bacterialspecies. Preferably, the prokaryotic microorganism is an attenuatedstrain of Salmonella. However, other prokaryotic microorganisms, such asattenuated strains of Escherichia coli, Shigella, Yersinia,Lactobacillus, Mycobacteria, Listeria or Vibrio are likewisecontemplated for the invention. Examples of suitable strains ofmicroorganisms include, but are not limited to, Salmonella typhimurium,Salmonella Typhi, Salmonella dublin, Salmonella enteritidis, Escherichiacoli, Shigella flexneri, Shigella sonnei, Vibrio cholerae (Yamamoto, etal. (2009) Gene 438:57-64), Pseudomonas aeruginosa (Lesic, et al. (2008)BMC Mol Biol 9:20), Yersinia pestis (Sun, et al. (2008) Applied and EnvMicrobiol 74:4241-4245), and Mycobacterium bovis (BCG). Of note, lambdared does not work in mycobacteria, but another phage (for example, Che9cgp61) has been used for recombineering in mycobacteria (van Kessel, etal. (2008) Methods mol biol 435:203-215).

In one preferred embodiment the prokaryotic microorganism is Salmonellatyphi Ty21a. VIVOTIF® Typhoid Vaccine Live Oral Ty21a is a liveattenuated vaccine intended for oral administration. The vaccinecontains the attenuated strain Salmonella Typhi Ty21a. (Germanier et al.(1975) J. Infect. Dis., 131:553-558). It is manufactured by Bema BiotechLtd. Berne, Switzerland. Salmonella Typhi Ty21a is also described inU.S. Pat. No. 3,856,935.

The attenuated strain of the prokaryotic microorganism undergoesrecombineering, such that a large antigenic region is integrated intothe chromosome of the microorganism. In one embodiment, the largeantigenic region is a Shigella sonnei O-antigen biosynthetic generegion, and the prokaryotic organism is a Salmonella Typhi Ty21a.

In a further aspect, the present invention provides a compositioncomprising one or more of above attenuated prokaryotic microorganisms,optionally in combination with a pharmaceutically or physiologicallyacceptable carrier. Preferably, the composition is a vaccine, especiallya vaccine for mucosal immunization, e.g., for administration via theoral, rectal, nasal, vaginal or genital routes. Advantageously, forprophylactic vaccination, the composition comprises one or more strainsof Salmonella expressing a plurality of different O-Ps genes or ofdifferent protein antigens (e.g. anthrax protective antigen or malariaparasite surface proteins).

In a further aspect, the present invention provides an attenuated strainof a prokaryotic microorganism described above for use as a medicament,especially as a vaccine.

In a further aspect, the present invention provides the use of anattenuated strain of a prokaryotic microorganism comprising an O-antigenbiosynthetic gene region inserted into a chromosome of the prokaryoticmicroorganism, wherein the O antigens are produced in the microorganism,in the preparation of a medicament for the prophylactic or therapeutictreatment of one or several different types of bacterial infection (e.g.typhoid fever and dysentery).

Generally, the microorganisms expressing O-Ps according to the presentinvention are provided in an isolated and/or purified form, i.e.,substantially pure. This may include being in a composition where itrepresents at least about 90% active ingredient, more preferably atleast about 95%, more preferably at least about 98%. Such a compositionmay, however, include inert carrier materials or other pharmaceuticallyand physiologically acceptable excipients. A composition according tothe present invention may include in addition to the microorganismsexpressing O-Ps as disclosed, one or more other active ingredients fortherapeutic or prophylactic use (e.g. other antigens) and an adjuvant.

The compositions of the present invention are preferably given to anindividual in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwho/what is being treated. Prescription of treatment, e.g., finaldecisions on acceptable dosage etc., will be dictated by VaccineRegulatory Authorities, after review of safety and efficacy datafollowing human immunizations.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically or physiologically acceptableexcipient, carrier, buffer, stabilizer or other materials well known tothose skilled in the art. Such materials should be non-toxic and shouldnot interfere with the efficacy of the active ingredient. The precisenature of the carrier or other material will depend on the route ofadministration.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (Ed.), 1980.

The invention further relates to the identification and sequencing of 9ORFs in the rfb locus (GENBANK® accession number: AY585348; SEQ ID NO:3)and an ORF in the rfp locus (GENBANK® accession number: AY763519; SEQ IDNO:4) for S. dysenteriae 1 LPS. These genes may be present in whole orin part in the vaccine strains described herein.

Accordingly, the present invention relates to vaccine strains furthercharacterized by the presence of heterologous genes or a set ofheterologous genes coding for O-Ps.

In a preferred embodiment of the vaccine strains, the heterologousgene(s) is (are) stably integrated into the chromosome of said strain ata defined integration site which is to be nonessential for growth, forinducing a protective immune response by the carrier strain.

In one embodiment, the heterologous genes of the invention include all 9ORFs from the rfb locus and the ORF from rfp. In another embodiment, theninth ORF from rfb is not present, because it is not essential for O-Psbiosynthesis. The ORFs may, in one embodiment, be separated by aninsertion sequence element. The latter is the case for S. sonnei only.

The ORFs may be under the control of the cognate promoter or othernon-cognate promoters. The essential O-antigen biosynthetic genes may bephysically together in one operon or separated on the chromosome andpresent on separate DNA regions under the control of differentpromoters. The genes may vary in gene order when placed next to oneanother, as long as they are biologically functional.

Alternatively, the above vaccine strains contain the rfbB, rfbC, andrfbA and/or any additional gene(s) necessary for the synthesis ofcomplete core-linked O-antigen LPS which are integrated in tandem into asingle chromosomal site or located at separate chromosomal sites.

Such vaccine strains allow expression of heterologous O-Ps, which iscovalently coupled to a heterologous LPS core region, which, preferably,exhibits a degree of polymerization similar to that of native LPSproduced by the enteric pathogen. Such vaccine strains can, if desired,be modified in such a way that they are deficient in the synthesis ofhomologous LPS core.

The invention also relates to a live vaccine comprising the abovevaccine strain and optionally a pharmaceutically or physiologicallyacceptable carrier and/or a buffer for neutralizing gastric acidityand/or a system for delivering said vaccine in a viable state to theintestinal tract.

Said vaccine comprises an immunoprotective or immunotherapeutic andnon-toxic amount of said vaccine strain. Suitable dosage amounts can bedetermined by the person skilled in the art and are typically 10⁷ to10¹⁰ bacteria.

Pharmaceutically and physiologically acceptable carriers, suitableneutralizing buffers, and suitable delivering systems can be selected bythe person skilled in the art.

In a preferred embodiment said live vaccine is used for immunizationagainst gram-negative enteric pathogens.

The mode of administration of the vaccines of the present invention maybe any suitable route which delivers an immunoprotective orimmunotherapeutic amount of the vaccine to the subject. However, thevaccine is preferably administered orally or intranasally.

The invention also relates to the use of the above vaccine strains forthe preparation of a live vaccine for immunization against gram-negativeenteric pathogens. For such use the vaccine strains are combined withthe carriers, buffers and/or delivery systems described above.

The invention also provides polypeptides and correspondingpolynucleotides required for synthesis of core linked O-specificpolysaccharide. The invention includes both naturally occurring andunnaturally occurring polynucleotides and polypeptide products thereof.Naturally occurring O-antigen biosynthesis products include distinctgene and polypeptide species as well as corresponding species homologsexpressed in organisms other than Shigella or Salmonella strains.Non-naturally occurring O-antigen biosynthesis products include variantsof the naturally occurring products such as analogs and O-antigenbiosynthesis products, which include covalent modifications. Thesequences of the O-antigen biosynthesis polynucleotides include SEQ IDNOs:3-4 (DNA including Shigella dysenteriae serotype 1 polypeptides),which are disclosed in U.S. Pat. No. 8,071,113.

Purified and isolated Shigella sonnei polynucleotides (e.g., DNAsequences and RNA transcripts, both sense and complementary antisensestrands) encode the bacterial O-antigen biosynthesis gene products.Certain DNA sequences, including genomic and cDNA sequences as well aswholly or partially chemically synthesized DNA sequences, are describedin U.S. Pat. No. 8,071,113. Genomic DNA comprises the protein codingregion for a polypeptide of the invention and includes variants that maybe found in other bacterial strains of the same species. “Synthesized,”as used herein and is understood in the art, refers to purely chemical,as opposed to enzymatic, methods for producing polynucleotides. “Wholly”synthesized DNA sequences are therefore produced entirely by chemicalmeans, and “partially” synthesized DNAs embrace those wherein onlyportions of the resulting DNA were produced by chemical means. PreferredDNA sequences encoding Shigella sonnei O-antigen biosynthesis geneproducts are set out in SEQ ID NO:2, and species homologs thereof.Preferred DNA sequences encoding Shigella dysenteriae O-antigenbiosynthesis gene products are set out in SEQ ID NOs:3 and 4, andspecies homologs thereof.

Autonomously replicating recombinant expression constructions such asplasmid and viral DNA vectors incorporating O-antigen biosynthesis genesequences are also provided. Expression constructs wherein O-antigenbiosynthesis polypeptide-encoding polynucleotides are operatively linkedto an endogenous or exogenous expression control DNA sequence and atranscription terminator are also provided. The O antigen biosynthesisgenes may be cloned by PCR, using Shigella sonnei genomic DNA as thetemplate. For ease of inserting the gene into expression vectors, PCRprimers are chosen so that the PCR-amplified gene(s) has a restrictionenzyme site at the 5′ end preceding the initiation codon ATG, and arestriction enzyme site at the 3′ end after the termination codon TAG,TGA or TAA. If desirable, the codons in the gene(s) are changed, withoutchanging the amino acids, according to E. coli codon preferencedescribed by Grosjean et al. (1982) Gene, 18:199-209; and Konigsberg etal. (1983) Proc. Natl. Acad. Sci. USA, 80:687-691. Optimization of codonusage may lead to an increase in the expression of the gene product whenproduced in E. coli. If a protein gene product is to be producedextracellularly, either in the periplasm of E. coli or other bacteria,or into the cell culture medium, the gene is cloned into an expressionvector and linked to a signal sequence.

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, either stably or transientlytransformed, transfected, or electroporated with polynucleotidesequences of the invention in a manner which permits expression of Oantigen biosynthesis polypeptides of the invention. Potential expressionsystems of the invention include bacterial, yeast, fungal, viral,parasitic, invertebrate, and mammalian cells systems. Host cells of theinvention are a valuable source of immunogen for development ofanti-bodies specifically immunoreactive with the O antigen. Host cellsof the invention are conspicuously useful in methods for large scaleproduction of O antigen biosynthesis polypeptides wherein the cells aregrown in a suitable culture medium and the desired polypeptide productsare isolated from the cells or from the medium in which the cells aregrown by, for example, immunoaffinity purification or any of themultitude of purification techniques well known and routinely practicedin the art. Any suitable host cell may be used for expression of thegene product, such as E. coli, other bacteria, including P. multocida,Bacillus and S. aureus, yeast, including Pichia pastoris andSaccharomyces cerevisiae, insect cells, or mammalian cells, includingCHO cells, utilizing suitable vectors known in the art. Proteins may beproduced directly or fused to a peptide or polypeptide, and eitherintracellularly or extracellularly by secretion into the periplasmicspace of a bacterial cell or into the cell culture medium. Secretion ofa protein requires a signal peptide (also known as pre-sequence); anumber of signal sequences from prokaryotes and eukaryotes are known tofunction for the secretion of recombinant proteins. During the proteinsecretion process, the signal peptide is removed by signal peptidase toyield the mature protein.

To simplify the protein purification process, a purification tag may beadded either at the 5′ or 3′ end of the gene coding sequence. Commonlyused purification tags include a stretch of six histidine residues (U.S.Pat. Nos. 5,284,933 and 5,310,663), a streptavidin affinity tagdescribed by Schmidt et al. (1993) Protein Eng., 6:109-122, a FLAGpeptide (Hopp et al. (1988) Biotechnology, 6:1205-1210), glutathione5-transferase (Smith et al. (1988) Gene, 67:31-40), and thioredoxin(LaVallie et at. (1993) Bio/Technology, 11:187-193). To remove thesepeptide or polypeptides, a proteolytic cleavage recognition site may beinserted at the fusion junction. Commonly used proteases are factor Xa,thrombin, and enterokinase.

In one embodiment, the invention employs purified and isolated Shigellasonnei O-antigen biosynthesis polypeptides as described above. Presentlypreferred are polypeptides comprising the amino acid sequences encodedby any one of the polynucleotides set out in SEQ ID NO:2, and specieshomologs thereof. In certain embodiments, the invention utilizes Oantigen biosynthesis polypeptides encoded by a DNA selected from thegroup consisting of:

a) the DNA sequence set out in any one of SEQ ID NO:2 and specieshomologs thereof;

b) DNA molecules encoding Shigella sonnei O-antigen biosyntheticpolypeptides encoded by any one of SEQ ID NO:2, and species homologsthereof; and

c) a DNA molecule encoding a O-antigen biosynthesis gene product thathybridizes under moderately stringent conditions to the DNA of (a) or(b). Moderately stringent hybridization conditions are well-known to theordinarily skilled artisan.

The invention also embraces polypeptides that have at least about 99%,at least about 95%, at least about 90%, at least about 85%, at leastabout 80%, at least about 75%, at least about 70%, at least about 65%,at least about 60%, at least about 55%, and at least about 50% identityand/or homology to the preferred polypeptides of the invention. Percentamino acid sequence “identity” with respect to the preferredpolypeptides of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the O antigen biosynthesis gene product sequence afteraligning both sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Percentsequence “homology” with respect to the preferred polypeptides of theinvention is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in one ofthe O antigen biosynthesis polypeptide sequences after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity. Conservativesubstitutions can be defined as set out below in Tables A and B.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G, A, P I, L, V Polar-uncharged C, S, T, M N, QPolar-charged D, E K, R Aromatic H, F, W, Y Other N, Q, D, E

Polypeptides of the invention may be isolated from natural bacterialcell sources or may be chemically synthesized, but are preferablyproduced by recombinant procedures involving host cells of theinvention. O antigen biosynthesis gene products of the invention may befull length polypeptides, biologically active fragments, or variantsthereof which retain specific biological or immunological activity. Thebiological activity is, in one embodiment, the biosynthesis of theantigen, for example, the Shigella sonnei O-antigen. The immunologicalactivity is, in one embodiment, the protective immunological activity ofthe antigenic gene product, for example, the O-antigen product. Variantsmay comprise O antigen biosynthesis polypeptide analogs wherein one ormore of the specified (i.e., naturally encoded) amino acids is deletedor replaced or wherein one or more non-specified amino acids are added:(1) without loss of one or more of the biological activities orimmunological characteristics (activity) specific for the O antigenbiosynthesis gene product; or (2) with specific disablement of aparticular biological activity of the O antigen biosynthesis geneproduct. Deletion variants contemplated also include fragments lackingportions of the polypeptide not essential for biological activity, andinsertion variants include fusion polypeptides in which the wild-typepolypeptide or fragment thereof have been fused to another polypeptide.

Variant O antigen biosynthesis polypeptides include those whereinconservative substitutions have been introduced by modification ofpolynucleotides encoding polypeptides of the invention. Conservativesubstitutions are recognized in the art to classify amino acidsaccording to their related physical properties and can be defined as setout in Table A (from WO 1997/009433, page 10). Alternatively,conservative amino acids can be grouped as defined in Lehninger,(Biochemistry, Second Edition (1975) W.H. Freeman & Co., pp. 71-77) asset out in Table B.

TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A, L, I, V, P B. Aromatic: F,W C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl:S, T, Y B. Amides: N, Q C. Sulfhydryl: C D. Borderline: G PositivelyCharged (Basic): K, R, H Negatively Charged (Acidic): D, E

Variant O antigen biosynthesis products of the invention include matureO antigen biosynthesis gene products, i.e., wherein leader or signalsequences are removed, having additional amino terminal residues. Oantigen biosynthesis gene products having an additional methionineresidue at position −1 are contemplated, as are O antigen biosynthesisproducts having additional methionine and lysine residues at positions−2 and −1. Variants of these types are particularly useful forrecombinant protein production in bacterial cell types. Variants of theinvention also include gene products wherein amino terminal sequencesderived from other proteins have been introduced, as well as variantscomprising amino terminal sequences that are not found in naturallyoccurring proteins.

The invention also embraces variant polypeptides having additional aminoacid residues which result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as fusion protein with glutathione-S-transferase (GST)provide the desired polypeptide having an additional glycine residue atposition −1 following cleavage of the GST component from the desiredpolypeptide. Variants which result from expression using other vectorsystems are also contemplated.

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, humanized, human, and CDR-grafted antibodies, includingcompounds which include CDR sequences which specifically recognize apolypeptide of the invention) and other binding proteins specific for Oantigen biosynthesis gene products or fragments thereof. The term“specific for” indicates that the variable regions of the antibodies ofthe invention recognize and bind O antigen exclusively (i.e., areoftentimes able to distinguish a single O antigen from related Oantigens, but may also interact with other proteins (for example, S.aureus protein A or other antibodies in ELISA techniques) throughinteractions with sequences outside the variable region of theantibodies, and in particular, in the constant region of the molecule.Screening assays to determine binding specificity of an antibody of theinvention are well known and routinely practiced in the art. For acomprehensive discussion of such assays, see Harlow et al. (Eds.);Antibodies A Laboratory Manual (1988) Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y., chapter 6. Antibodies that recognize and bindfragments of the O antigen of the invention are also contemplated,provided that the antibodies are first and foremost specific for, asdefined above, an O antigen of the invention from which the fragment wasderived.

Treatment/Therapy

In certain embodiments, the present invention provides compositions(including vaccines) and methods to treat (e.g., alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more symptoms or features of) and/orprevent bacterial infection.

In some embodiments, methods of vaccination and/or treatment (such asthose described in the sections below) involve stratification of apatient population based on prior exposure to bacterial strains. Suchmethods involve steps of determining whether a patient has beenpreviously exposed to one or more of the bacterial strains. In someembodiments, if it is determined that a patient has been previously beenexposed to one or more of the bacterial strains, that patient mayreceive less concentrated, less potent, and/or less frequent doses ofthe inventive vaccine or composition. If it is determined that a patienthas not been previously been exposed to one or more of the bacterialstrains, that patient may receive more concentrated, more potent, and/ormore frequent doses of the inventive vaccine or composition.

In one embodiment, the recombineered bacterial strain or vaccine orcomposition of the invention treats more than one bacterial infection,i.e., infection with more than one bacterial species. It can, in such anembodiment, be deemed a “multifunctional” vaccine (or strain orcomposition—either of the latter two are contemplated in the continueddescription, below). A multifunctional vaccine according to theinvention may also be useful for treating and/or preventingsimultaneously a number of different disorders in a subject.Accordingly, the present invention further provides, in an additionalembodiment, a method for treating and/or preventing more than onedisorder in a subject, by administering to the subject a multifunctionalvaccine according to the invention.

Exemplary disorders which potentially may be treated and/or prevented bya multifunctional vaccine according to the invention include, withoutlimitation, multiple bacterial species infections, burns, infections,neoplasia, or radiation injuries. “Neoplasia” refers to the uncontrolledand progressive multiplication of tumour cells, under conditions thatwould not elicit, or would cause cessation of, multiplication of normalcells. Neoplasia results in a “neoplasm”, which is defined herein tomean any new and abnormal growth, particularly a new growth of tissue,in which the growth of cells is uncontrolled and progressive. Thus,neoplasia includes “cancer”, which herein refers to a proliferation oftumour cells having the unique trait of loss of normal controls,resulting in unregulated growth, lack of differentiation, local tissueinvasion, and/or metastasis.

Administration

Compositions and vaccines (the terms used interchangeably herein) may beadministered using any amount and any route of administration effectivefor treatment and/or vaccination. The exact amount required will varyfrom subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. Compositions are typically formulated in dosage unit form for easeof administration and uniformity of dosage. It will be understood,however, that the total daily usage of the compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular subject or organism will depend upon a varietyof factors including the disorder being treated and/or vaccinated andthe severity of the disorder; the activity of the specific vaccinecomposition employed; the half-life of the composition afteradministration; the age, body weight, general health, sex, and diet ofthe subject; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors, well known in the medical arts.

Compositions (and vaccines) according to the invention may beadministered by any route. In some embodiments, pharmaceuticalcompositions of the present invention are administered by a variety ofroutes, including oral (PO), intravenous (IV), intramuscular (IM),intra-arterial, intramedullary, intrathecal, subcutaneous (SQ),intraventricular, transdermal, interdermal, intradermal, rectal (PR),vaginal, intraperitoneal (IP), intragastric (IG), topical ortranscutaneous (e.g., by powders, ointments, creams, gels, lotions,and/or drops), mucosal, intranasal, buccal, enteral, vitreal,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/orthrough a portal vein catheter.

In general, the most appropriate route of administration will dependupon a variety of factors including the nature of the agent beingadministered (e.g., its stability upon administration), the condition ofthe subject (e.g., whether the subject is able to tolerate a particularmode of administration), etc. In specific embodiments, compositions maybe administered intranasally. In specific embodiments, compositions maybe administered by intratracheal instillation. In specific embodiments,compositions may be administered by bronchial instillation. In specificembodiments, compositions may be administered by inhalation. In specificembodiments, compositions may be administered as a nasal spray. Inspecific embodiments, compositions may be administered mucosally. Inspecific embodiments, compositions may be administered orally. Inspecific embodiments, compositions may be administered by intravenousinjection. In specific embodiments, compositions may be administered byintramuscular injection. In specific embodiments, compositions may beadministered by subcutaneous injection. The oral or nasal spray oraerosol route (e.g., by inhalation) is most commonly used to delivertherapeutic agents directly to the lungs and respiratory system.However, the invention encompasses the delivery of a pharmaceuticalcomposition by any appropriate route taking into consideration likelyadvances in the sciences of drug delivery.

For oral administration, the composition (including vaccine) may bepresented as capsules, tablets, dissolvable membranes, powders,granules, or as a suspension. The composition may have conventionaladditives, such as lactose, mannitol, corn starch, or potato starch. Thecomposition also may be presented with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch, or gelatins.Additionally, the composition may be presented with disintegrators, suchas corn starch, potato starch, or sodium carboxymethylcellulose. Thecomposition may be further presented with dibasic calcium phosphateanhydrous or sodium starch glycolate. Finally, the composition may bepresented with lubricants, such as talc or magnesium stearate.

For parenteral administration, the composition may be combined with asterile aqueous solution, which is preferably isotonic with the blood ofthe subject. Such a formulation may be prepared by dissolving a solidactive ingredient in water containing physiologically-compatiblesubstances, such as sodium chloride, glycine, and the like, and having abuffered pH compatible with physiological conditions, so as to producean aqueous solution, then rendering said solution sterile. Theformulation may be presented in unit or multi-dose containers, such assealed ampoules or vials. The formulation also may be delivered by anymode of injection, including any of those described herein.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, dissolvable membranes, and granules. In such soliddosage forms, the active ingredient is mixed with at least one inert,pharmaceutically acceptable excipient such as sodium citrate ordicalcium phosphate and/or fillers or extenders (e.g., starches,lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g.,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents(e.g., agar, calcium carbonate, potato starch, tapioca starch, alginicacid, certain silicates, and sodium carbonate), solution retardingagents (e.g., paraffin), absorption accelerators (e.g., quaternaryammonium compounds), wetting agents (e.g., cetyl alcohol and glycerolmonostearate), absorbents (e.g., kaolin and bentonite clay), andlubricants (e.g., talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), taste/olfactorycomponents, and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

Dosage forms for topical and/or transdermal administration of acomposition in accordance with this invention may include ointments,pastes, creams, lotions, gels, powders, solutions, sprays, inhalantsand/or patches. Generally, the active ingredient is admixed understerile conditions with a pharmaceutically acceptable excipient and/orany needed preservatives and/or buffers as may be required.Additionally, the present invention contemplates the use of transdermalpatches, which often have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms may be prepared,for example, by dissolving and/or dispensing the compound in the propermedium. Alternatively or additionally, the rate may be controlled byeither providing a rate controlling membrane and/or by dispersing thecompound in a polymer matrix and/or gel.

For transdermal administration, the composition (including vaccine)according to the invention may be combined with skin penetrationenhancers, such as propylene glycol, polyethylene glycol, isopropanol,ethanol, oleic acid, N-methylpyrrolidone, and the like, which increasethe permeability of the skin to the composition, and permit thecomposition to penetrate through the skin and into the bloodstream. Thecomposition of enhancer and vaccine also may be further combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which may be dissolved in solvent, such asmethylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch. The composition may beadministered transdermally, at or near the site on the subject where theinfection, neoplasm, or other disorder may be localized. Alternatively,the composition may be administered transdermally at a site other thanthe affected area, in order to achieve systemic administration.

For intranasal administration (e.g., nasal sprays) and/or pulmonaryadministration (administration by inhalation), a composition (includingvaccine) according to the invention, including aerosol formulations, maybe prepared in accordance with procedures well known to persons of skillin the art. Aerosol formulations may comprise either solid particles orsolutions (aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers,ultrasonic nebulizers, etc.) and atomizers may be used to produceaerosols from solutions (e.g., using a solvent such as ethanol);metered-dose inhalers and dry-powder inhalers may be used to generatesmall-particle aerosols. The desired aerosol particle size can beobtained by employing any one of a number of methods known in the art,including, without limitation, jet-milling, spray drying, andcritical-point condensation.

Compositions for intranasal administration may be solid formulations(e.g., a coarse powder) and may contain excipients (e.g., lactose).Solid formulations may be administered from a container of powder heldup to the nose, using rapid inhalation through the nasal passages.Compositions for intranasal administration may also comprise aqueous oroily solutions of nasal spray or nasal drops. For use with a sprayer,the formulation may comprise an aqueous solution and additional agents,including, for example, an excipient, a buffer, an isotonicity agent, apreservative, or a surfactant. A nasal spray may be produced, forexample, by forcing a suspension or solution of the composition througha nozzle under pressure.

Formulations of a composition (including vaccine) according to theinvention for pulmonary administration may be presented in a formsuitable for delivery by an inhalation device, and may have a particlesize effective for reaching the lower airways of the lungs or sinuses.For absorption through mucosal surfaces, including the pulmonary mucosa,the formulation may comprise an emulsion that includes, for example, abioactive peptide, a plurality of submicron particles, a mucoadhesivemacromolecule, and/or an aqueous continuous phase. Absorption throughmucosal surfaces may be achieved through mucoadhesion of the emulsionparticles.

Compositions (including vaccines) according to the invention for usewith a metered-dose inhaler device may include a finely-divided powdercontaining the composition as a suspension in a non-aqueous medium. Forexample, the composition may be suspended in a propellant with the aidof a surfactant (e.g., sorbitan trioleate, soya lecithin, or oleicacid). Metered-dose inhalers typically use a propellant gas (e.g., achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon) stored in a container (e.g., a canister) as a mixture(e.g., as a liquefied, compressed gas). Inhalers require actuationduring inspiration. For example, actuation of a metering valve mayrelease the mixture as an aerosol. Dry-powder inhalers usebreath-actuation of a mixed powder.

A composition (including vaccine) according to the invention also may bereleased or delivered from an osmotic mini-pump or other timed-releasedevice. The release rate from an elementary osmotic mini-pump may bemodulated with a microporous, fast-response gel disposed in the releaseorifice. An osmotic mini-pump would be useful for controlling release,or targeting delivery, of the composition.

A composition (including vaccine) according to the invention may beadministered or introduced to a subject by known techniques used for theintroduction of drugs, including, for example, injection andtransfusion. Where a disorder is localized to a particular portion ofthe body of the subject, it may be desirable to introduce thecomposition directly to that area by injection or by some other means(e.g., by introducing the composition into the blood or another bodyfluid).

A composition (including vaccine) according to the invention may beadministered to a subject who has a disorder, either alone or incombination with one or more drugs used to treat that disorder. Forexample, where the subject has neoplasia, the composition may beadministered to a subject in combination with at least oneantineoplastic drug. Examples of antineoplastic drugs with which thecomposition may be combined include, without limitation, carboplatin,cyclophosphamide, doxorubicin, etoposide, and vincristine. Additionally,when administered to a subject who suffers from neoplasia, thecomposition may be combined with other neoplastic therapies, including,without limitation, surgical therapies, radiotherapies, gene therapies,and immunotherapies.

Vaccine

A “vaccine” is a composition that induces an immune response in therecipient or host of the vaccine. The vaccine can induce protectionagainst infection upon subsequent challenge with a bacterial species (orother microorganism). Protection refers to resistance (e.g., partialresistance) to persistent infection of a host animal with at least onebacterial species (or other microorganism). Neutralizing antibodiesgenerated in the vaccinated host can provide this protection. In othersituations, CTL responses can provide this protection. In somesituations, both neutralizing antibodies and cell-mediated immune (e.g.,CTL) responses provide this protection.

Vaccines are useful in preventing or reducing infection or disease byinducing immune responses, to an antigen or antigens, in an individual.For example, vaccines can be used prophylactically in naive individuals,or therapeutically in individuals already infected with at least onebacterial species (or other microorganism).

Protective responses can be evaluated by a variety of methods. Forexample, either the generation of neutralizing antibodies againstbacterial (or other microorganism) proteins, and/or the generation of acell-mediated immune response against such proteins can indicate aprotective response. Protective responses also include those responsesthat result in lower number of bacteria colonized in a vaccinated hostanimal exposed to a given inoculum (of bacteria or other microorganism)as compared to a host animal exposed to the same inoculum, but that hasnot been administered the vaccine.

Vaccines according to the invention may, in one embodiment, contain anadjuvant. The term “adjuvant”, as used herein, refers to any compoundwhich, when injected together with an antigen, non-specifically enhancesthe immune response to that antigen. Exemplary adjuvants includeComplete Freund's Adjuvant, Incomplete Freund's Adjuvant, Gerbu adjuvant(GMDP; C.C. Biotech Corp.), RIBI fowl adjuvant (MPL; RIBI ImmunochemicalResearch, Inc.), potassium alum, aluminum phosphate, aluminum hydroxide,QS21 (Cambridge Biotech), TITERMAX® adjuvant (CytRx Corporation), andQUIL A® adjuvant. Other compounds that may have adjuvant propertiesinclude binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, or gelatin; excipients such as starch,lactose or dextrins, disintegrating agents such as alginic acid, sodiumalginate, PRIMOGEL®, corn starch and the like; lubricants such asmagnesium stearate or STEROTEX®; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

Furthermore, a useful compendium of many adjuvants is prepared by theNational Institutes of Health and can be found on the internet(www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf). Hundreds ofdifferent adjuvants are known in the art and could be employed in thepractice of the present invention. Exemplary adjuvants that can beutilized in accordance with the invention include, but are not limitedto, cytokines, aluminum salts (e.g., aluminum hydroxide, aluminumphosphate, etc.), gel-type adjuvants (e.g., calcium phosphate, etc.);microbial adjuvants (e.g., immunomodulatory DNA sequences that includeCpG motifs; endotoxins such as monophosphoryl lipid A); exotoxins suchas cholera toxin, E. coli heat labile toxin, and pertussis toxin;muramyl dipeptide, etc.); oil-emulsion and emulsifier-based adjuvants(e.g., Freund's Adjuvant, MF59 [Novartis], SAF, etc.); particulateadjuvants (e.g., liposomes, biodegradable microspheres, etc.); syntheticadjuvants (e.g., nonionic block copolymers, muramyl peptide analogues,polyphosphazene, synthetic polynucleotides, etc.); and/or combinationsthereof. Other exemplary adjuvants include some polymers (e.g.,polyphosphazenes), Q57, saponins (e.g., QS21), squalene,tetrachlorodecaoxide, CPG 7909, poly[di(carboxylatophenoxy)phosphazene](PCCP), interferon-gamma, block copolymer P1205 (CRL1005), interleukin-2(IL-2), polymethyl methacrylate (PMMA), etc. In one embodiment of theinstant invention, the carrier bacterium (e.g. S. Typhi Ty21a) itselfserves as an adjuvant for expressed foreign antigens such as Shigella Oantigens or anthrax protective antigen.

Vaccines according to the invention may, in another embodiment, beformulated using a diluent. Exemplary “diluents” include water,physiological saline solution, human serum albumin, oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents,antibacterial agents such as benzyl alcohol, antioxidants such asascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates and agents for adjusting the osmolarity, such as sodiumchloride or dextrose. Exemplary “carriers” include liquid carriers (suchas water, saline, culture medium, saline, aqueous dextrose, and glycols)and solid carriers (such as carbohydrates exemplified by starch,glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified byascorbic acid and glutathione, and hydrolyzed proteins.

Vaccines according to the invention may, in still another embodiment,contain an excipient. The term “excipient” refers herein to any inertsubstance (e.g., gum arabic, syrup, lanolin, starch, etc.) that forms avehicle for delivery of an antigen. The term excipient includessubstances that, in the presence of sufficient liquid, impart to acomposition the adhesive quality needed for the preparation of pills ortablets.

As mentioned above, in some embodiments, interfering agents and/orbinding agents in accordance with the invention may be utilized forprophylactic applications. In some embodiments, prophylacticapplications involve systems and methods for preventing, inhibitingprogression of, and/or delaying the onset of bacterial infection. Insome embodiments, interfering agents may be utilized for passiveimmunization (i.e., immunization wherein antibodies are administered toa subject). In some embodiments, vaccines for passive immunization maycomprise antibody interfering agents, such as those described herein. Insome embodiments, passive immunization occurs when antibodies aretransferred from mother to fetus during pregnancy. In some embodiments,antibodies are administered directly to an individual (e.g., byinjection, orally, etc.). Of note, it is possible to immunize a personand use their antibodies for passive protection in another individual.For example, Ty21a is contraindicated for use in pregnant women (i.e.,they are immunocompromised, and the attenuated Salmonella couldpotentially cause illness). However, it may be appropriate to passivelytransfer protective antibodies to a pregnant mother, instead of leavingthem susceptible to deadly diseases.

The invention provides, in one embodiment, vaccines for activeimmunization (i.e., immunization wherein microbes, proteins, peptides,epitopes, mimotopes, etc. are administered to a subject). In someembodiments, the vaccines may comprise one or more interfering agentsand/or binding agents, as described herein.

In one embodiment, a composition is provided including interferingagents and/or binding agents, as described for vaccines, above. Forexample, in some embodiments, interfering agent and/or binding agentpolypeptides, nucleic acids encoding such polypeptides, characteristicor biologically active fragments of such polypeptides or nucleic acids,antibodies that bind to and/or compete with such polypeptides orfragments, small molecules that interact with or compete with suchpolypeptides or with glycans that bind to them, etc. are included incompositions. In some embodiments, interfering agents and/or bindingagents that are not polypeptides, e.g., that are small molecules,umbrella topology glycans and mimics thereof, carbohydrates, aptamers,polymers, nucleic acids, etc., are included in the compositions. Onecould, in specific embodiments, envision bacterially delivered therapiesfor cancer or other maladies, in which the therapeutic nucleic acids orproteins are encoded in the chromosome of the carrier bacterialvaccine/therapeutic strain.

The invention encompasses treatment and/or prophylaxis of bacterial (orother microorganism) infections by administration of compositionsaccording to the invention. In some embodiments, such compositions areadministered to a subject suffering from or susceptible to a bacterialinfection. In some embodiments, a subject is considered to be sufferingfrom a bacterial infection if the subject is displaying one or moresymptoms commonly associated with the bacterial infection. In someembodiments, the subject is known or believed to have been exposed tothe at least one bacterial species (or other microorganism). In someembodiments, a subject is considered to be susceptible to a bacterialinfection if the subject is known or believed to have been exposed tothe bacterial species. In some embodiments, a subject is known orbelieved to have been exposed to the bacterial species if the subjecthas been in contact with other individuals known or suspected to havebeen infected with the same, and/or if the subject is or has beenpresent in a location in which the bacterial infection is known orthought to be prevalent. Compositions provided herein may beadministered prior to or after development of one or more symptoms ofbacterial (or other microorganism) infection.

In general, a composition will include a “therapeutic agent” (therecombineered Salmonella Typhi Ty21a, for example), in addition to oneor more inactive, agents such as a sterile, biocompatible pharmaceuticalcarrier including, but not limited to, sterile water, saline, bufferedsaline, or dextrose solution. Alternatively or additionally, acomposition may comprise a pharmaceutically acceptable excipient, which,as used herein, includes any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,disintegrating agents, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, buffering agents, solidbinders, granulating agents, lubricants, coloring agents, sweeteningagents, flavoring agents, perfuming agents, and the like, as suited tothe particular dosage form desired. Remington's The Science and Practiceof Pharmacy, 21st Ed., A. R. Gennaro, (Lippincott, Williams & Wilkins,Baltimore, Md., 2006; incorporated herein by reference) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. Except insofar as anyconventional excipient medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any other componentof the pharmaceutical composition, its use is contemplated to be withinthe scope of this invention.

Combinations

Compositions and vaccines according to the invention can be administeredto a subject either alone or in combination with one or more othertherapeutic agents including, but not limited to, vaccines and/orantibodies. By “in combination with,” it is not intended to imply thatthe agents must be administered at the same time or formulated fordelivery together, although these methods of delivery are within thescope of the invention. Compositions and vaccines according to theinvention can be administered concurrently with, prior to, or subsequentto, one or more other desired therapeutics or medical procedures. Itwill be appreciated that therapeutically active agents utilized incombination may be administered together in a single composition oradministered separately in different compositions. In general, eachagent will be administered at a dose and/or on a time scheduledetermined for that agent.

In general, each agent (in this context, one of the “agents” is acomposition or vaccine according to the invention) will be administeredat a dose and on a time schedule determined for that agent.Additionally, the invention encompasses the delivery of the compositionsin combination with agents that may improve their bioavailability,reduce or modify their metabolism, inhibit their excretion, or modifytheir distribution within the body. Although the compositions (includingvaccines) according to the invention can be used for treatment and/orvaccination of any subject, they are preferably used in the treatmentand/or vaccination of humans.

The particular combination of therapies (e.g., therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will be appreciated thatthe therapies employed may achieve a desired effect for the same purpose(for example, an agent useful for treating, preventing, and/or delayingthe onset of a bacterial (or other microorganism) infection may beadministered concurrently with another agent useful for treating,preventing, and/or delaying the onset of the bacterial infection), orthey may achieve different effects (e.g., prevention of severe illnessor control of adverse effects).

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

Dosages

The dosage of a vaccine (or other composition) according to theinvention can be determined by, for example, first identifying doseseffective to elicit a prophylactic and/or therapeutic immune response.This may be accomplished by measuring the serum titer ofbacterium-specific immunoglobulins and/or by measuring the inhibitoryratio of antibodies in serum samples, urine samples, and/or mucosalsecretions. The dosages can be determined from animal studies, includinganimals that are not natural hosts to the bacterial species in question.For example, the animals can be dosed with a vaccine candidate, e.g., avaccine according to the invention, to partially characterize the immuneresponse induced and/or to determine if any neutralizing antibodies havebeen produced. In addition, routine human clinical studies can beperformed to determine the effective dose for humans.

Effective doses may be extrapolated from dose-response curves derivedfrom in vitro and/or in vivo animal models. Ty21a is given every otherday for three or four doses (depending on country of use), and the dosemay be between about 2×10⁹ and >10¹⁰ colony forming units (cfu) perdose. In one embodiment, dose spacing of every one to two months worksbetter in some immunization schedules (e.g. in infants), and in anotherembodiment, doses as high as 10¹¹ cfu may work better in developingcountries (i.e., trigger better protection that persists longer).

An immunologically effective amount, based upon human studies, would, inone embodiment, be sufficient to stimulate an acceptable level ofprotective immunity in a population. For some vaccines (in certainembodiments), this immunologically effective level would provide an 80%efficacy against a specific diarrheal disease. For other vaccines (inother embodiments), an immunologically effective amount would be onethat protects against severe disease but may not protect against allsymptoms of a disease.

In one embodiment, a vaccine (or other composition) according to theinvention may also be administered to a subject at risk of developing adisorder, in an amount effective to prevent the disorder in the subject.As used herein, the phrase “effective to prevent the disorder” includeseffective to hinder or prevent the development or manifestation ofclinical impairment or symptoms resulting from the disorder, or toreduce in intensity, severity, and/or frequency, and/or delay of onsetof one or more symptoms of the disorder.

Kits

Kits comprising the recombineered Salmonella Typhi Ty21a or a vaccine ora composition according to the invention are provided in an additionalembodiment. Kits can include one or more other elements including, butnot limited to, instructions for use; other reagents, e.g., a diluent,devices or other materials for preparing the vaccine or composition foradministration; pharmaceutically acceptable carriers; and devices orother materials for administration to a subject. Instructions for usecan include instructions for therapeutic application (e.g., DNAvaccination and protein boosting) including suggested dosages and/ormodes of administration, e.g., in a human subject, as described herein.

In another embodiment, a kit according to the invention can furthercontain at least one additional reagent, such as a diagnostic ortherapeutic agent, e.g., a diagnostic agent to monitor an immuneresponse to the compositions or vaccines according to the invention inthe subject, or an additional therapeutic agent as described herein(see, e.g., the section herein describing combination therapies).

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1. Cloning the Minimal Essential S. sonnei O-AntigenBiosynthetic Genes

A large 30 kb region encoding S. sonnei form I O-antigen biosynthesisand flanking sequences were previously characterized (initially bydeletion analysis) to determine minimal necessary sequences forO-antigen expression. Together with DNA sequence analyses of thisregion, these deletion data indicated that a contiguous 12.3 kb regioncontaining a putative promoter and 10 orfs (orf 4 to 13 in FIG. 1) wasrequired for O-antigen biosynthesis in S. sonnei. These deletion studiesalso suggested that wzz (ofr3), typically involved in regulatingO-antigen chain length, is not essential for form 1 expression in Ty21a,but the latter part of wzz apparently contains the putative form 1O-antigen biosynthetic operon promoter (Xu, et al. (2002) Infect andImmun 70:4414-4423.

Construction of pMD-TV Plasmid

Standard molecular biology techniques were used for cloning. Therestriction endonucleases and ligase were purchased from New EnglandBiolabs (NEB) or FERMENTAS®, and PHUSION® polymerase (Fisher) was usedfor all PCRs. The Kan^(r) cassette flanked by FRT sites wasPCR-amplified from pKD4 with primers containing NheI and NsiIrestriction sites, and plasmid pGB-2 was also PCR amplified with primerscontaining NheI and NsiI restriction sites, engineered to delete theopen reading frame (orf) that confers Spc resistance. The two PCRproducts were digested with NheI and NsiI and ligated to construct theKan^(r), Spc^(S) plasmid pMD35-36. The vexA gene (˜1000 bp) and part ofthe tviD gene (˜500 bp) were PCR-amplified from Ty21a genomic DNA. Theamplified tviD sequence was cloned upstream of the Kan^(r) cassetteflanked by FRT sites, and the vexA amplicon was cloned downstream of themultiple cloning site (MCS), as shown in FIG. 1. Restrictionendonuclease sites were further added or removed, as needed, by themethod described in the PHUSION® site-directed mutagenesis kit (NEB), toconstruct pMD-TV. Sequences of the primers used to construct pMD-TV areprovided in Table C, below.

TABLE C Primers used to construct pMD-TV Primer name Sequence^(a)prMD31.pGB.2.F.Nsi TCATATATGCATTAATGTCTAACAATTCGTTCAAGCC (SEQ. ID NO: 5)prMD32.pGB2.R.NheI CTACACGCTAGCACGCTACTTGCATTACAGCTTACG (SEQ. ID NO: 6)prMD33.KD.F.NheI TAGCGTGCTAGCGTGTAGGCTGGAGCTGCTTCG (SEQ. ID NO: 7)prMD34.KD.R.NsiI ACATTAATGCATATATGAATATCCTCCTTAGTTCCTATTCCG (SEQ. ID NO: 8) prMD35.pGB.MCS.F.pTCGAGGGGCGCGCCGGTACCGAATTCCCGACAGTAAGA CGG (SEQ. ID NO: 9)prMD36.pGB.MCS.R.p GCCCGGGGATCCGCGGCCGCGTCGACCTGCAGCCAAGCTT (SEQ. ID NO: 10) prMD58.tviD.FGATCGGCTAGCCCGTTCCTCATTGATTTGATTGC (SEQ. ID NO: 11) prMD59.tviD.RGATCGCCCGGGTTACGACTTCCCTGATGTATTTTTTTGT AATG (SEQ. ID NO: 12)prMD60.vexA.F GATCGCTCGAGTAATTAATGGGCATCATTTTTCAGCTATTTC (SEQ. ID NO: 13) prMD83.vexA.R.xhoI.1000GATCGCTCGAGTTAGAAAGAATTAGTGCCGCGGG (SEQ. ID NO :14) prMD75.mcs.F.pCGGTCTCGAGGGGCGCGCCGGTACCGAATTCTAATTAATGGGCATCATTTTTCAGCTATTTC (SEQ. ID NO: 15) prMD76.mcs.R.pGTGGATCCAAGCTTGCGGCCGCCCGTCGACAGTAAAGCCCTCGCTAGATTTTAATGCG (SEQ. ID NO: 16) prMD77.sites.remove.F.pCCGACAGTAAGACGGGTAAGCC (SEQ. ID NO: 17) prMD78.sites.remove.R.pTTAGAAAGAATTAGTGCCGCGGG (SEQ. ID NO: 18) ^(a)Restriction sites areunderlined.

Part of wzz (orf 3) through orf 7 was PCR-amplified (labeled fragment Ain FIG. 1) and cloned into pMD-TV.

Cloning S. sonnei O-Antigen Genes into pMD-TV

S. sonnei O-antigen genes were PCR-amplified from pXK65 (Xu et al.(2002) Infect. Immun., 70:4414-4423). Primers prMD43.ss.F.HindIII,gatcaaagcttgatcaaatagctcatat tcagcg (SEQ ID NO:19), andprMD44.ss.BamHI.R, gatcaggatcctgctcagtccggttggtg (SEQ ID NO:20), wereused to amplify part of the wzz gene (orf3) through wzy (orf7), shown asfragment A in FIG. 1. The resulting PCR product and pMD-TV were digestedwith HindIII and BamHI, PCR purified (QIAGEN®) and ligated using T4ligase. Primers prMD41.ss.F.BamHI, gtagcggatccaagcgcagctatttaggatgag(SEQ ID NO:21), and prMD42.ss.R.XhoI,gatcgctcgagttaatttacggggtgattaccagac (SEQ ID NO:22), were used toamplify genes wbgV (orf9) through apqZ (orf 14), shown as fragment B inFIG. 1. The resulting PCR product and plasmid pMD-TV containing geneswzz to wzy (orf3 to orf7) were digested with BamHI and XhoI,PCR-purified, and ligated as before to construct pMD-TV-Ss-1. Thus, orfs9 to 14 (labeled fragment B in FIG. 1) were PCR-amplified, eliminatingthe non-essential IS630 element (orf8), and this amplicon was attachedto cloned fragment A in pMD-TV to generate pMD-TV-Ss-1.

The plasmid was observed to express S. sonnei form I O-antigen in eitherE. coli or Ty21a, as determined by slide agglutination with formI-specific antisera. Also, LPS purified from E. coli or Ty21a carryingpMD-TV-Ss-1 reacted with specific form I antibody in Westernimmunoblotting studies (FIG. 2), demonstrating that IS630 (orf8) is notessential for form I O-antigen biosynthesis.

Example 2. Integration into the Ty21a Chromosome of Linear Shigella DNA

Datsenko and Wanner (Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA,97:6640-6645) previously described a method based on the highlyefficient λ phage red recombination system that enables one to createspecific targeted gene mutations via recombinational replacement of anE. coli chromosomal sequence with a small selectable antibioticresistance gene that is generated by PCR using primers containing 36 to50 bp extensions that are homologous to the gene targeted for mutation.The λ red system includes β, γ, and exo genes, whose products are calledBeta, Gam, and Exo, respectively (Murphy (1998) J. Bacteriol.,180:2063-2071). Gam inhibits the host RecB,C,D exonuclease and theSbcC,D nuclease activities, so that exogenously added linear DNA is notdegraded. The Exo protein is a dsDNA-dependent exonuclease that binds tothe terminus of each strand while degrading the other strand in a 5′ to3′ direction. Beta binds to the resulting ssDNA overhangs, ultimatelypairing them with a complementary chromosomal DNA target (Sawitzke etal. (2007) Methods Enzymol., 421:171-199). The low copy plasmid pKD20encodes these three λ red proteins under the control of thearabinose-inducible P_(araB) promoter, has an optimized ribosome-bindingsite for efficient translation of the β, γ, and exo genes, and has atemperature-sensitive replicon to allow for its easy elimination(Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA, 97:6640-6645). The λred system has been widely utilized for specific gene inactivation in E.coli, Salmonella and Shigella species, and for introducing smallbiological tags (Uzzau et al. (2001) Proc. Natl. Acad. Sci. USA,98:15264-15269) or single genes into these chromosomes (Yu et al. (2011)Appl. Microbiol. Biotechnol., 91:177-188).

In this study, the lengths of the homologous extensions needed fordetectable lambda-Red mediated recombination of large blocks of foreignDNA (≧15,000*bp) into a targeted site in the desired chromosome wereincreased in a stepwise fashion from 50 to 150 bp and then to 500-1000bp. In order to integrate the large form 1 O-antigen operon into thechromosome of vaccine strain Ty21a, a new plasmid, pMD-TV, wasconstructed.

As depicted in FIG. 1, the optimal chromosome insertion vector pMD-TVconsists of a replicon plus a multiple cloning site (MCS) and anadjacent selectable Kan^(r) cassette, which can be removed, whendesired, via special recombination between flanking FRT sites. Inaddition, the MCS plus removable Kan^(r) cassette are flanked by twolarge regions (500-1000 bp) of targeted homology with the Ty21achromosome. The homologous regions chosen are tviD (˜500 bp) and vexA(1000 bp) located within the non-expressed Vi capsule locus of Ty21a.The minimal essential S. sonnei O-antigen gene region was cloned intothe MCS of pMD-TV to construct pMD-TV-Ss-1, the PCR template forsubsequent chromosome recombineering. The resulting PCR amplicon,containing the S. sonnei O-antigen genes, is 15,532 bp.

Bacterial Strains, Plasmids, and Growth Conditions

The bacterial strains and plasmids utilized herein are described inTable D, below. E. coli strains were grown at 37° C. in Luria-Bertani(LB) broth or on LB agar from DIFCO®. Shigella strains were grown intryptic soy broth (TSB) or tryptic soy agar (TSA) from DIFCO®.Salmonella strain Ty21a was grown in TSB or TSA supplemented with 0.01%galactose and 0.01% glucose. Plasmid-containing strains were selected ingrowth medium containing ampicillin (Amp; 100 μg/ml), spectinomycin(Spc; 100 μg/ml), chloramphenicol (Cm; 35 μg/ml), or kanamycin (Kan; 30μg/ml). All constructed plasmids were sequenced, and the sequences wereassembled and analyzed by using the VECTOR NTI® suite 9.0 software(Invitrogen).

TABLE D Bacterial strains and plasmids Reference Strain or plasmidGenotype or description or source Strains E. coli DH5α supE44 hsdR17recA1 endA1 New England gyrA96 thi-1 relA1 Biolabs S. enterica serovargalE ilvD viaB (Vi-) H₂S- (Germanier Typhi Ty21a and Fuer, 1975) S.flexneri 2a 2457 Lab stock S. flexneri 3a J99 Lab stock Ty21a-Ss (MD77)S. sonnei O-antigen genes integrated This study into tviD-VexA on thechromosome, Kan^(s), Amp^(s), Cm^(s) Ty21a-Ss-Kan S. sonnei O-antigengenes integrated This study (MD67) into tviD-VexA on the chromosome,Kan^(r), Amp^(s), Cm^(s) Ty21a-Sd (MD149) S. dysenteriae O-antigen genesThis study, integrated into tviD-VexA on the FIG. 4 chromosome,expressed from lpp promoter, Kan^(s), Amp^(s), Cm^(s) Ty21a-Sd1 S.dysenteriae O-antigen genes This study (MD174) integrated into tviD-VexAon the FIG. 5 chromosome, expressed from lpp promoter, Kan^(s), Amp^(s),Cm^(s) Ty21a-Y (MD114) S. flexneri rfb operon integrated into This studytviD-vexA on the chromosome, FIG. 14 expressed from native promoter,Kan^(s), Amp^(s), Cm^(s) Ty21a-2a (MD194) S. flexneri 2a O-antigen genesThis study integrated into tviD-vexA on the FIG. 14 chromosome,expressed from native promoter, Kan^(s), Amp^(s), Cm^(s) Ty21a-3a(MD196) S. flexneri 3a O-antigen genes This study integrated intotviD-vexA on the FIG. 14 chromosome, expressed from native promoter,Kan^(s), Amp^(s), Cm^(s) Ty21a-2a1 (MD212) S. flexneri 2a O-antigengenes This study integrated into tviD-vexA on the FIG.3 14 chromosome,expressed from lpp promoter, Kan^(s), Amp^(s), Cm^(s) S. dysenteriaetype 1 Virulent strain (Mendizabal- 1617 Morris et al., 1971; Neill etal., 1988) 1617ΔstxA Deletion of most of stxA of strain (Xu et al., 16172007) S. sonnei 53GI Form I (phase I), virulent isolate (Kopecko et al.,1980) Plasmids pGB-2 pSC101 derivative, low-copy (Churchward plasmid;Sm^(r), Spc^(r) et al., 1984) pKX65 S. sonnei genes cloned into pGB-2(Xu et al., 2002) pXK65 pGB-2 containing the cloned Rfb (Xu et al.,region of S. sonnei 2002 pKD4 Kan^(r) flanked by FRT sites, Amp^(r),(Datsenko oriR6Kgamma and Wanner, 2000) pKD46 Gam-beta-exo proteinsunder the (Datsenko control of arabinose promoter, ts- and Wanner,rep^(a), Amp^(r) 2000) pCP20 yeast Flp recombinase gene FLP, (CherepanovCm^(r), Amp^(r), ts-rep^(a) and Wackernagel, 1995) pMD35-36 pSC101derivative, low-copy This study plasmid, Kan^(r) flanked by FRT pMD-TVpSC101 derivative, low-copy This study plasmid, Kan^(r) flanked by FRT,containing homologous regions for Ty21a tviD and vexA genes pMD-TV-Ss-1S. sonnei O-antigen genes cloned This study (SEQ ID NO: 24) into pMD-TVpMD-TV-Sd-4 S. dysenteriae 1 O-antigen genes This study cloned intopMD-TV pMD-TV-lpp lpp promoter cloned into pMD-TV This study ^(a)ts-rep;temperature sensitive replicationChromosomal Integration of O-Antigen Genes

The S. sonnei O-antigen biosynthetic genes described above, engineeredbetween 500-1000 bp regions of Ty21a chromosome homology to enhancerecombination efficiency, were integrated into the Ty21a chromosomeusing λ red recombination (Datsenko et al. (2000) Proc. Natl. Acad. Sci.USA, 97:6640-6645). Ty21a was transformed with pKD46 and grown at 30° C.in TBS-Amp supplemented with 0.01% galactose and 0.01% glucose andinduced with L-arabinose until OD₆₀₀=˜0.7. The resulting cells were madeelectrocompetent by washing with 10 mM Hepes and 10% glycerol andconcentrating 500-fold by centrifugation. As shown in FIG. 1,pMD-TV-Ss—was used as a template with the tviD forward primer and vexAreverse primer for PCR amplification. This PCR-amplified region containspart of the tviD gene, a Kan^(r) gene flanked by FTR sites, agenetically-minimized set of essential S. sonnei O-antigen biosyntheticgenes, and the vexA gene.

The PCR product was DpnI-digested to remove circular plasmid DNA, waspurified, and ˜1-2 μg was transformed into freshly grownelectrocompetent cells of Ty21a expressing λ red proteins. Thus, thelinear ds-DNA amplicon was used to transform competent Ty21a cellsexpressing the λ red proteins from pKD46.

Transformants for the above-described amplicon were selected forKan^(r); the transformed cells were recovered by growing in SOC media at30° C. and plating on TSA-Kan plates. Of note, the above PCR productswere DpnI-digested to remove any remaining circular plasmids. Despitethis step, a few copies of plasmid inevitably escaped DpnI-digestion andgenerated Kan^(r) transformants. These plasmid transformants can thenrecombine at either chromosomal region of homology, creating unstablemerodiploids, making molecular characterization confusing, because PCRresults reflect a mixture of possibilities. After primary selection withantibiotic, mutants were maintained on medium without an antibiotic.Isolates were colony-purified once non-selectively at 37° C. and thentested for ampicillin sensitivity to demonstrate loss of plasmid pKD46.

To detect appropriate chromosomal integrants (i.e., containing 15,532 kbinserts of S. sonnei form 1 genes inserted between tviD and vexA), DNAfrom individual transformants was subjected to PCR using primers prMD92and prMD124 (FIG. 1) recognizing Ty21a sequences just upstream anddownstream of the targeted integration site. Colonies were furtheranalyzed by PCR to confirm chromosomal integration with primers prMD92,GTTGCGGTAATGGTATAACGAAATAACAGATAC (SEQ ID NO:25) and prMD124,CACGCAATATTTCAATGATGGCAAC (SEQ ID NO:26), as shown in FIG. 1.Furthermore, expression of the form 1 O-antigen was confirmed byslide-agglutination and Western immunoblot with form 1-specificantisera. (DIFCO®-BBL). The wild-type Ty21a sequence, in the integrationregion tviD-tviE-vexA, detected by using primers prMD92 and prMD124,generated a ˜4 kb band. Undesired integration of the whole plasmid intothe Ty21a chromosome at tviD and/or vexA could also be detected by PCR.

Next, the desired Kan^(r) chromosomal integrants of linear S. sonnei DNAwere transformed with temperature-sensitive plasmid pCP20, whichexpresses the FLP recombinase, to eliminate the FRT-flanked Kan^(r)cassette. In brief, chromosomal integrants expressing Kan^(r) weretransformed with pCP20 (Cherepanov et al. (1995) Gene, 158:9-14), andCm^(r) transformants were selected at 30° C., after which a few isolateswere colony-purified non-selectively at 37° C. and then tested for lossof all antibiotic resistances.

Antibiotic-sensitive chromosomal integrants were further analyzed by PCRto demonstrate deletion of the Kan cassette. Furthermore, the final PCRproducts generated from primers prMD92 and prMD124, were sequencedentirely. When the chromosomally integrated Ty21a-Ss sequence wascompared to the GENBANK® S. sonnei O-antigen gene cluster AF294823 (SEQID NO:23), only a single change (E₂₇₃K) was detected in wbgZ (orf13 inFIG. 1), which had no apparent effect on O-antigen expression, asdetermined by slide agglutination and western blotting of resulting LPS.

Example 3. Two-Year Ear Studies to Attain Stable Expression of theApproximately 12 kb Shigella sonnei O-Antigen Gene Region ViaRecombinational Insertion into the Chromosome of Vaccine Vector StrainSalmonella Typhi Ty21a

Expression in Low Copy Plasmid Vector

S. sonnei O-antigen genes were cloned into the low copy plasmid pGB-2,and this heterologous O-antigen was stably expressed in vaccine vectorTy21a, with only a 2% loss after 60 generations. This plasmid contains aspectinomycin-resistance gene cassette for genetic selection purposes.In contrast, high copy plasmid vectors, containing this cloned Shigellaregion were not genetically stable (3). Although pGB-2 appeared suitablystable, the spectinomycin-resistance gene needed to be removed.

Achieving Stable Expression after Removal of Antibiotic Cassette

S. sonnei genes on pGB-2 were stably expressed with 98% stability for 60generations of growth in the absence of antibiotics. For FDA regulatorypurposes, however, the antibiotic resistance cassette needs to beremoved prior to administration of the vaccine to humans on a largescale. Thus, attempts were made to remove the resistance gene cassetteby deletion using targeted restriction endonucleases, by deletion usingrecombination techniques or PCR-generated deletions. Regardless of themethod used or the length of the region deleted, removal of theantibiotic cassette resulted, unexpectedly, in plasmid instability.Plasmid loss over 60 generations reached 50%. The spectinomycin cassettewas then replaced with a kanamycin-resistance gene cassette flanked byFRT sites. If the kanamycin-resistance could be removed viarecombination between FRT sites, perhaps the resulting plasmid would begenetically stable. However, removal of kanamycin-resistance alsoresulted in plasmid instability.

Construction of pMD35-36 for Chromosomal Integration of O-Antigen Genes

In order to achieve stable expression of heterologous Shigella sonneiO-antigen genes in Ty21a in the absence of an antibiotic resistancecassette, the O-antigen genes were integrated into the Ty21a chromosome.To facilitate this task, the low copy plasmid pMD35-36 was constructedto contain a kanamycin-resistance gene cassette, flanked by FRT sites,and located adjacent to a multiple cloning site (MCS) locus. Inaddition, the kanamycin-resistance cassette, FRT sites, and MCS locuswere synthesized by PCR from this plasmid using primers containingregions of homology to the Ty21a chromosome for recombinationalinsertion. The Ty21a tviD and vexA gene regions were used for homology.They are located adjacent to each other in the Vi capsule biosynthesisoperon, which is nonfunctional in Ty21a.

Cloning S. sonnei Genes into pMD35-36

Essential genes of the S. sonnei O-antigen operon (eliminating anon-essential IS630 element) were cloned into plasmid pMD35-36 in twosteps.

Optimizing Lambda Red Expression in Ty21a

Datsenko and Wanner (2000) (Datsenko et al. (2000) Proc. Natl. Acad.Sci. USA, 97:6640-6645) previously described a method based on thehighly efficient lambda phage red recombination system that enables oneto create mutations within a targeted gene via recombinationalreplacement of a chromosomal sequence with a selectable antibioticresistance gene that is generated by PCR, using primers with 36 to 50 bpextensions that are homologous to the gene targeted for mutation. Lambdared proteins are expressed from plasmid pKD46, and the expression istightly regulated through an arabinose-inducible promoter. This lambdared system has been widely utilized for specific gene inactivation in E.coli, Salmonella and Shigella species (Kotloff et al. (1999) Bull. WHO,77:651-666), and for introducing small biological tags (Kewon (2008)Curr. Opin. Infect. Dis., 21:313-318).

To facilitate chromosomal insertion, the S. sonnei O-antigen genes werecloned into plasmid pMD35-36, as described above. Initially, 50 bpregions of chromosomal homology to tviD and vexA were used. PCRamplification of this large recombinational construct resulted in a˜13.5 kb fragment, which includes the S. sonnei O-antigen biosyntheticgenes, the kanamycin-resistance gene cassette flanked by FRT sites, allcontained within two regions of chromosomal homology, consisting of 50bp homology to tviD and vexA. This PCR product was transformed intoTy21a cells containing pKD46. No anticipated chromosomal inserts,detected as kanamycin-resistant colonies, were obtained, even when thisexperiment was repeated. As an experimental control, attempts were madeto insert the smaller 1.5 kb kanamycin-resistance gene cassette, withoutany Shigella sequences included. This also resulted in failure. Thisresult was unexpected, as the smaller 1.5. kb Kan^(R) insert fragmentshould have integrated into the chromosome.

Based on a hypothesis that this lack of success might be due toinsufficient lamda red recombinant protein expression in SalmonellaTyphi Ty21a (the published conditions for lambda red expression (i.e., 1mM arabinose) in Salmonella (Kewon (2008) Curr. Opin. Infect. Dis.,21:313-318) and E. coli (Kotloff et al. (1999) Bull. WHO, 77:651-666)were not successful in Ty21a), higher arabinose concentrations of 2 mM,10 mM, or 25 mM were attempted and found to be insufficient to expresslambda red proteins from pKD46 in Ty21a, as detected by chromosomalintegration of the 1.5 kb kanamycin-resistance cassette. This result wasvery unexpected.

Finally, a 100-fold increase in the recommended 1 mM concentration ofarabinose was tried, and it was found that 100 mM was needed to generatesufficient lambda red expression in Ty21a to facilitate chromosomalinsertion of the small 1.5 kb kanamycin-resistance gene cassette, using50 bp regions of chromosomal homology. However, the large 13.5 kb PCRproduct containing the S. sonnei O-antigen biosynthetic genes was notintegrated into the chromosome.

Homology Extensions Increased to 150 Bases

In order to facilitate chromosomal integration of the larger insert, thechromosomal homology extensions on the PCR primers were increased from50 to 150 bases each. Although these increased regions of homologyfacilitated chromosomal insertion of the 1.5 kb kanamycin-resistancecassette at an enhanced frequency, the large 13.5 kb PCR product failedto integrate into the chromosome after several attempts. This result wasalso unexpected, since general recombination usually proceeds with50-100 bp of homology, and lambda Red-mediated recombination is usuallymore efficient than bacterial general recombination.

Construction of Chromosomal Insertion Plasmid pMD-TV

The use of large 500-1000 bp regions of homology required cloning theseregions into the chromosomal insertion plasmid. First, Ty21a genomic DNAwas prepared. Next, the vexA gene (˜1000 bp) and part of the tviD gene(˜500 bp) were PCR-amplified from Ty21a genomic DNA. These two PCRamplicons were cloned into pMD35-36 in separate steps. In addition, theMCS locus was further changed to contain new and different restrictionsites. This resulted in the construction of a new chromosomal insertionvector, pMD-TV, containing 500-1000 bp arms of chromosomal homology.These longer arms were engineered to increase the area of homology withthe Ty21a chromosome and thus increase the chances for integration oflarge DNA regions (i.e., greater than 5 kb).

Cloning S. sonnei Genes into pMD-TV

Essential genes of the S. sonnei O-antigen biosynthetic operon werecloned in two steps into the plasmid pMD-TV, eliminating a nonessentialIS630 element.

Optimizing Competent Cells

After the S. sonnei genes were cloned into the new pMD-TV plasmid, a15.5 kb PCR product, which contains the kanamycin-resistance cassette,the S. sonnei O-antigen genes, and the arms of Ty21a homology, wastransformed into Ty21a expressing the lambda red proteins. Unexpectedly,no transformants were achieved, even after exhaustive plating and arepeat experiment. In order to address the question of whether thetransformation efficiency of the large linear DNA fragment was too low,fresh competent Ty21a cells were further optimized by increasing thenumber of final washes from two to three, increasing the number of cellsin each transformation reaction, and also increasing the amount oflinear DNA added per reaction to about 1-2 μg. These changes allowed forthe successful chromosomal integration into Ty21a of the large 13.5 kbfragment encoding the heterologous S. sonnei O-antigen. (Datsenko et al.(2000) Proc. Natl. Acad. Sci. USA, 97:6640-6645; Uzzau et al. (2001)Proc. Natl. Acad. Sci. USA, 98:15264-15269; Xu et al. (2002) Infect.Immun., 70:4414-4423).

Example 4. O-Antigen Expression Determined by Silver Staining andImmunoblotting. LPS Analysis by Silver-Stain and Western Immunoblottingwas Employed to Examine LPS Expression in Parent and Recombinant StrainsExpressing Heterologous O-Antigen

O-Antigen Expression Analyses

Slide agglutination reactions were performed with rabbit polyclonalantisera against S. sonnei (phase I) or against Salmonella TyphiO-specific 9 factor (DIFCO®). For immunoblotting, Salmonella, Shigellaand E. coli strains with or without various recombinant plasmids weregrown overnight with aeration at 37° C. in media containing appropriateantibiotics. LPS was purified using an LPS extraction kit (BoccaScientific) according to the manufacturer's instructions. Purified LPSwas separated by Tris-glycine PAGE. Standard Western blotting procedureswere carried out with the above antibodies for identification ofspecific LPS types. Silver-staining analysis was performed using theSILVERXPRESS® Silver Staining Kit (Invitrogen) according to themanufacturer's instructions. As revealed by silver-staining of isolatedpolysaccharide separated by SDS-PAGE (FIG. 2, Panel A), E. coli DH5α, arough mutant (lane 1), was observed to express S. sonnei O-antigen asboth an LPS ladder and a slower-migrating Group 4 capsule (lane 2).Ty21a (lane 3) was found to produce a typical 9,12 LPS short ladderpattern, which is altered both by the addition of an extended form 1 LPSladder pattern and slower-moving form 1 O-antigen capsule expression(lanes 4, 5, 6, 7). Wild-type S. sonnei was observed to express a form 1LPS ladder pattern, the majority topping out at a chain length of ˜20O-repeats, by silver stain analysis (FIG. 2, Panel A, lane 8).

Western immunoblotting (FIG. 2, Panel B) showed specific antibodyreaction with form 1 polysaccharide in all strains expressing S. sonneiO-antigen, regardless of whether the form 1 genes were carried byplasmids (i.e., pXK65, pMD-TV-Ss-1) or integrated into the Ty21achromosome [i.e., Ty21a-Ss (or MD77) Ty21a-Ss-Kan (or MD67)]. Although aform 1 LPS ladder pattern was easily visible in E. coli (FIG. 2, PanelB, lane 2) and wild-type S. sonnei (lane 8), the majority of form 1O-antigen in all expressing isolates appeared to be the slower migratinggroup 4 capsule expression. As noted below, Form 1 O-antigen was highlyimmunogenic regardless of expression as LPS or in capsular form (FIG.3).

Example 5. Stability of Heterologous Form 1 O-Antigen Expression inTy21a

Stability of recombinant clones of Ty21a-Ss expressing S. sonneiO-antigen were tested by immunoblotting of colonies plated from anovernight culture (˜24 hours), and it was diluted 1:100 and grown for anadditional˜24 hours. Colonies, transferred to a nitrocellulose membrane,were analyzed by standard Western blotting procedures using theLPS-specific antibodies specified above.

The new recombineered vaccine strain Ty21a-Ss, which isantibiotic-sensitive and contains chromosomally integrated form 1biosynthetic genes, was grown at high dilution in TSB plus 0.01%galactose and 0.01% glucose without antibiotics for ˜24 hours, whichrepresents ˜25 generations of growth. This culture was diluted 1:100 andgrown for an additional ˜24 hours, which represents 50 generations. Theresulting cells were plated on TSA containing 0.01% galactose and 0.01%glucose, grown overnight at 37° C., and colony immunoblots wereperformed to examine form 1 expression, as described above. All of 500colonies tested retained S. sonnei form I O-antigen expression,demonstrating 100% stability of the chromosomally integrated genes.

Example 6. Antibodies to Both S. Typhi and S. sonnei LPSs are Elicitedin Mice Following Immunization with Ty21a-Ss

Mice were immunized (3 doses IP spaced 2 weeks apart) with therecombineered vaccine strain Ty21a-Ss, the parent strain Ty21a orcontrol PBS. Eight-week-old female Balb/c mice were immunized withvaccine candidate strains (Ty21a-Ss) or negative controls, Ty21a alone,and PBS. Ty21a-Ss and Ty21a controls were grown overnight in TSBsupplemented with 0.01% galactose and 0.01% glucose, washed, andsuspended in sterile PBS to a concentration of ˜0.8-1.6×10⁸ CFU per ml.They were tested one week after each dose for the presence of antibodiesagainst S. sonnei or Ty21a LPS by ELISA, as described below. As shown inFIG. 3, immunization with Ty21a-Ss resulted in increased anti-LPSantibody titers after each dose, but peaked after 2 doses at very hightiters of ˜400,000 for anti-form 1 serum IgG, and after 3 doses atsimilarly high titers for anti-Salmonella Typhi LPS antibodies. Theseresults further confirm the stable expression of both heterologous S.sonnei O-antigen and homologous Ty21a LPS in Ty21a-Ss. Moreover, theparent strain Ty21a elicited serum IgG antibodies that reacted only withTy21a LPS, and were at essentially the same titer as that elicited bythe recombinant Ty21a-Ss.

Example 7. Protection from Virulent S. sonnei Challenge Conferred byTy21a-Ss

Mice are typically used to demonstrate immune stimulation by aSalmonella Typhi vaccine strain and to measure specific protectionagainst parenteral challenge with virulent Shigella. Balb/C mice wereimmunized with Ty21a-Ss, Ty21a alone, or saline by the IP route withthree doses of vaccine spaced two weeks apart. Mice were inoculatedintraperitoneally with a 0.25-ml dose containing ˜2-4×10⁷ CFU per mouseof either vaccine, control Ty21a cells, or 0.25 ml sterile saline, forthree total doses spaced two weeks apart. Mice were tail-bled one weekafter every injection. Two weeks following the last dose, mice werechallenged i.p. with S. sonnei 53G at a dose of approximately 100×LD50.Immunized and control mice were challenged intraperitoneally, 2 weeksafter final immunization, with 5×10⁶ CFU/ml of freshly grown,mid-log-phase S. sonnei strain 53GI in 0.25 ml (2×10⁶ CFU per mouse) of5% hog gastric mucin (Sigma) dissolved in sterile saline (i.e.,approximately 100 times the 50% lethal infectious dose [LD₅₀]). Survivalwas monitored for 72 h.

TABFE E Mouse protection against virulent S. sonnei challenge Groups of10 mice immunized with: Survivors/total Ty21a-Ss (strain MD 77) 10/10 Ty21a 0/10 Saline 0/10Detection of Anti-LPS Antibodies by ELISA

S. sonnei 53G Phase 1 was grown overnight at 37° C. with aeration in TBSwhile S. Typhi Ty21a-Ss was grown in TSB supplemented with 0.01%galactose and 0.01% glucose. LPS was purified using a LPS extraction kit(Bocca Scientific) according to the manufacturer's instructions.Microtiter plates were coated with S. sonnei (phase I) or SalmonellaTyphi Ty21a purified LPS in 0.1 M Na₂CO₃/NaHCO₃ pH 9.5. Coatedmicrotiter plates were blocked with blocking buffer containing 1% BSA(Sigma) in TBST (TBS with 0.5% tween-20) for two hours. Serial dilutionsof serum were added to each plate and incubated at 4° C. overnight.After washing six times with TBST, bound antibodies were detected withHRP-conjugated goat anti-mouse IgG (SOUTHERNBIOTECH®). Endpoint titerswere defined as the reciprocal of the antibody dilution for the lastwell in a column with a positive OD for each sample after subtractingthe background. Background values were determined with pre-immunizationsera, where the OD values of the pre-immunization sera were averaged andthen doubled. This value was subtracted from the OD of all the wellscontaining titrations of every mouse serum sample. Each data pointrepresents the average endpoint titer of two independent ELISAsperformed for every mouse serum sample. The individual sample titers andthe mean±SEM for each group of ten mice are shown in FIG. 3.

Sequences are available under GENBANK® accession numbers JX436480 forpMD-TV (SEQ ID NO:1) and JX436479 for Ty21a-Ss tviD-vexA region (SEQ IDNO:2). This stringent challenge resulted in 100% mortality in Ty21aalone- or saline-immunized mice. However, mice immunized with therecombineered vaccine strain Ty21a-Ss were 100% protected against the S.sonnei challenge.

Thus, disclosed herein are attempts to stabilize the expression ofheterologous S. sonnei LPS antigens in oral vaccine vector Ty21a tocreate a candidate that will protect against both typhoid fever andshigellosis due to S. sonnei. Previous studies had shown that a clonedlarge block of Shigella LPS biosynthetic genes (i.e., 10-15 kb) was muchmore stable in the low copy plasmid pGB-2 than in high copy plasmidvectors (Xu et al. (2007) Vaccine, 25:6167-6175; Xu et al. (2002)Infect. Immun., 70:4414-4423). When attempting to remove the antibioticresistance selective marker in pGB-2, which removal would be requiredfor human vaccine use, unexpected and unexplained genetic instabilitywas encountered. For example, Kan^(r) (kanamycin resistance) pGB-2carrying the S. sonnei form 1 O-antigen biosynthetic genes was 95-98%genetically stable when grown for 60 generations in the absence ofantibiotic selective pressure. However, removal of the antibioticresistance gene function by insertion or small deletion resulted in aplasmid with ˜60% genetic stability (unpublished data).

A recent study by Yu, et al. (2011) inserted small ˜2 kb gene regionsinto the Salmonella chromosome. Thus, the λ red recombination system wasmodified to allow the introduction of large blocks of foreign DNA, morethan 15 kb, into the chromosome of Salmonella Typhi, termed genomicsuper-recombineering. This task required expanding the size of thenecessary homologous recombination regions from ˜50 bp as originallyproposed (Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA,97:6640-6645) to 500-1000 bp in order to increase the efficiency ofrecombination. These expanded regions of homology were engineered intoplasmid vector pMD-TV, which was specifically created to insert largeregions of DNA into a susceptible chromosome at a chosen, targetedintegration site (i.e., in this case, the tviE gene region of thenon-functional Vi gene locus of Ty21a was chosen). Furthermore, theexpression of the λ red recombination proteins in Ty21a and theconcentration of PCR-amplified DNA needed for efficient chromosomalinsertion of this large amplicon had to be optimized. Utilizing thisoptimized genomic super-recombineering technique, the S. sonneiO-antigen genes were inserted into the Ty21a Vi gene locus. HeterologousS. sonnei O-antigen expression in this Ty21a-Ss chromosomal integrant is100% genetically stable. Additionally, this method allows for the use ofantibiotic selection during strain construction and removal of aFRT-bracketed antibiotic resistance gene after all genetic manipulationsare completed. Of especial importance, this new vaccine candidate strainelicits robust serum IgG antibody responses against both heterologous S.sonnei form I LPS and homologous Salmonella Typhi LPS in mice andprotects mice 100% against a lethal challenge dose of virulent S.sonnei.

This chromosomal super-recombineering method has allowed for thedevelopment of a much improved vaccine candidate that is 100%genetically stable. This technique can be applied to many differentbacterial genera and can be used to insert different foreign antigens atmultiple targeted chromosomal sites, after which the associatedantibiotic resistance gene, used for efficient selection, can beremoved. The cloning of the S. dysenteriae serotype 1 O-antigenessential biosynthetic genes on plasmid pGB-2 has been previouslydescribed (Xu et al. (2007) Vaccine, 25:6167-6175). The pGB-2 cloning ofthe S. flexneri 2a and 3a LPS regions has been undertaken to constructadditional multivalent typhoid-shigellosis vaccine components. It is,thus, contemplated to make similar super-recombineered chromosomalinsertions of, for example, minimal-sized LPS genes of S. flexneri 2a,S. flexneri 3a, and S. flexneri 6, each into a separate Ty21a strain.The final multivalent vaccine will consist of 5 different strainscomprising a multifunctional vaccine for protection against entericfevers (aimed at typhoid, but with moderate cross-protection againstparatyphoid fevers) and the predominant causes of shigellosis, projectedto protect against ˜85% of shigellosis worldwide (Noriega et al. (1999)Infect. Immun., 67:782-788). In an additional embodiment, other antigensare incorporated into Ty21a (or another suitable vector strain) forvaccine purposes (e.g. anthrax protective antigen, plague F1 and Vantigens, viral antigens to protect against viral diseases, malariaantigens) or genetic constructs expressing anti-tumor proteins or siRNAsdirected at inhibiting tumor growth and metastasis for anti-cancertherapeutic purposes.

Recent collaborative studies (Ohtake et al. (2011) Vaccine,29:2761-2771) have led to the development of a formulation andfoam-drying method that results in a temperature-stable, dried Ty21avaccine that obviates the need for refrigeration duringdistribution/immunization and will extend the shelf life to 5 years orgreater at 4° C., enhancing the potential value of this multifunctionalenteric vaccine for use in travellers, the military, and in developingworld populations.

Example 8. Cloning and Chromosomal Insertion of a Second LPS Locus—ofShigella dysenteriae O-Antigen Biosynthetic Genes

Cloning S. dysenteriae O-Antigen Genes into pMD-TV

S. dysenteriae O-antigen genes were PCR-amplified from S. dysenteriaeserotype 1 strain 1617 genomic DNA (Xu, et al. (2007) Vaccine25:6167-6175). Primers prMD127.dy.F.HindIIIgatcgaagcttggcattttttgtcatttttggatgc (SEQ ID NO:27) andprMD128.dy.R.wbbP.BamHI gatcgggatccatcgatatggctgggtaaggtcatg (SEQ IDNO:28) were used first to amplify the wbbP gene. The resulting PCRproduct and pMD-TV were digested with HindIII and BamHI, PCR-purified,and ligated. Next, primers prMD129.F.BamHI gatcgggatcctaatgaaaatctgaccgaatgtaacgg (SEQ ID NO:29) and prMD130.R.EcoRIgatcggaattctcacattaatgctaccaaaaagagtcgc (SEQ ID NO:30) were used toamplify the larger S. dysenteriae 1 rfb gene cluster. The resulting PCRproduct and plasmid pMD-TV-wbbP were digested with BamHI and EcoRI,PCR-purified, and ligated to construct pMD-TV-Sd-4.

Integrating S. dysenteriae O-Antigen Genes into the Ty21a Chromosome

The S. dysenteriae O-antigen biosynthetic genes that were cloned intoplasmid pMD-TV to construct pMD-TV-Sd-4 were used as a template for PCRwith the tviD forward primer and vexA reverse primer. The PCR-amplifiedregion was 13,574 bp and contains part of the tviD gene, Kan^(r) genecassette flanked by FRT sites, S. dysenteriae O-antigen biosyntheticgenes and the vexA gene. The PCR amplicon was transformed into Ty21acompetent cells expressing λ red proteins from pKD46 and selected forKan resistance as described (Dharmasena et al.). PCR with primersupstream and downstream of the site of integration, prMD92 and prMD124,resulted in a ˜14.5 Kb Kan^(r) insert band. After removing the Kan^(r)cassette as previously described (Dharmasena et al.),antibiotic-sensitive chromosomal integrants were further analyzed by PCRfor deletion of the Kan^(r) cassette, and the PCR product (MD149/Ty21a-Sd˜13 Kb band) obtained from primers prMD92 and prMD124 wassequenced. The previously sequenced S. dysenteriae wpp gene (GENBANK®accession no. AY763519; SEQ ID NO:4) and that of the S. dysenteriaestrain 1617 strain were identical except for two bases, where CA ischanged to AC at position 619-620 in the strain 1617 wpp gene. Nomutations were detected in the chromosomally inserted wpp in Ty21a-Sdstrain. When the Ty21a-Sd chromosomal insert Rfb sequence was comparedto the GENBANK® accession no. AY585348 (SEQ ID NO:3) for the S.dysenteriae 1617 strain Rfb O-antigen gene cluster, only a single A to Gtransition (N130D) was detected in the gene rmlD (FIG. 4), which did notaffect O-antigen expression as determined by slide agglutination andELISA expression studies.

Replacing the Wpp Native Promoter with lpp

The low copy plasmid pMD-TV is a pSC 101 derivative existing as ˜5copies per cell. Thus, the plasmid pMD-TV-Sd-4 in Ty21a has ˜5 copies ofS. dysenteriae O-antigen genes per cell, while Ty21a-Sd (MD149) [i.e.,the strain with S. dysenteriae O-antigen genes integrated into the Ty21achromosome] has only one copy of the S. dysenteriae O-antigenbiosynthetic genes per cell. Note, from previous studies (Dharmasena, etal.), that the S. sonnei LPS:S. Typhi Ty21a LPS ratio, as assessed byELISA, was approximately 1 in strains expressing the S. sonnei O-antigengenes from the Ty21a chromosome (Ty21a-Ss) compared to the same genesexpressed from the pGB-2 plasmid (pMD-TV-Ss) in Ty21a. However, the S.dysenteriae LPS: S. Typhi Ty21a LPS ratio was considerably less than 1in the strain expressing S. dysenteriae O-antigen genes from thechromosome (Ty21a-Sd, MD 149) than from the plasmid (pMD-TV-Sd-4 inTy21a), as determined by ELISA. Thus, attempts were made to increase theS. dysenteriae LPS expression by replacing the native wpp promoter withthe Escherichia coli lpp promoter, which is a highly transcribedconstitutively active promoter.

pMD-TV-lpp was used as a template for PCR with the tviD forward primerand the lpp promoter reverse primer containing a 150 bp wpp homologyextension (prMD151.lpp.R

aggaatggcgcctgctttttttattatttccttggatgaatcattatagtcagtagcaaaagcatagactgaaatccctttcttggttagtgtttttattaaatccaatctaaacaaaatcatagcatttgctgtgttccctattattga gatcttcat atgcctctcctttcattattaataccctctagagttc ; SEQ ID NO: 31).The PCR-amplified region (which was 13,574 bp) contains part of the tviDgene, Kan^(r) cassette flanked by FTR sites, a 200 bp lpp promoter andthe first 150 bp of the S. dysenteriae 1 wpp gene. The PCR products weretransformed into Ty21a-Sd (MD149) competent cells expressing λ redproteins from pKD46 and selected initially for Kan^(r) resistance.Subsequently, the Kan^(r) cassette was removed and the remainingchromosomal insert was sequenced as described (Dharmasena, et al. 2013Intl J Med Microbiol 303:105-113) to construct Ty21a-Sdl (MD174).Expression data suggest that Ty21a-Sdl (MD174) expresses a moredesirable S. dysenteriae LPS: S. Typhi Ty21a LPS ratio of approximately1, than Ty21a-Sd (MD 149) containing the native promoter, as determinedby ELISA. Indeed, Western blot with anti-S. dysenteriae antibody showsthat replacing wpp native promoter with lpp (Ty21a-sdl, MD174) resultsin higher S. dysenteriae LPS expression than that of Ty21a-sd (MD149)and the LPS levels are comparable to plasmid expression (FIG. 6). AlsoO-antigen expression of both these strains were 100% stable over 75generations of growth in vitro determined by colony immunoblotting.Genetic Stability of Heterologous S. dysenteriae 1 O-Antigen Expressionin Ty21a

The newly recombineered vaccine strains Ty21a-Sd (MD149), SEQ ID NO:32,and Ty21-Sdl (MD174), SEQ ID NO:33, which are antibiotic-sensitive andcontain chromosomally integrated Sd1 O-antigen biosynthetic genes, weregrown at high dilution in Tryptic Soy broth (TSB) plus 0.01% galactoseand 0.01% glucose without antibiotics for ˜24 hours. This represents ˜25generations of growth. The resulting cells were plated on TSA containing0.01% galactose and 0.01% glucose, grown overnight at 37° C., and colonyimmunoblots were performed to examine S. dysenteriae O-antigenexpression with rabbit polyclonal antisera against S. dysenteriae 1(Difco). All of 200 colonies tested retained S. dysenteriae serotype 1O-antigen expression, demonstrating 100% stability of the chromosomallyintegrated genes

Mouse Immunogenicity

Eight week-old female AJ mice, 10 in each group, were immunized with twovaccine candidate strains (Ty21a-sd and Ty21a-sdl) or negative controlsTy21a alone and PBS. Ty21a-sd, Ty21a-sdl, and Ty21a controls were grownovernight in TSB supplemented with 0.01% galactose and 0.01% glucose,washed, and suspended in sterile PBS to a concentration of ˜4-8×10⁷ CFUper ml. Mice were inoculated intraperitoneally with a 0.5-ml dosecontaining ˜2-4×10⁷ CFU per mouse of either vaccine, control Ty21acells, or 0.5 ml sterile saline, for three total doses spaced two weeksapart. Mice were tail-bled one week after the last injection.Immunization with both vaccine strains (Ty21a-sd and Ty21a-sdl) resultedin a moderate antibody titer of around 15,000 against S. dysenteriaecompared to 400,000 for S. sonnei. Moreover, the parent strain Ty21aelicited serum IgG antibodies that cross reacted with S. dysenteriae LPSat a low level (˜500) (FIG. 7). Although Ty21a-sd elicited high antibodytiter against S. Typhi LPS (˜100,000), which was essentially the sametiter as that elicited by the parent strain Ty21a, the S. Typhi LPSantibody titer elicited by Ty21a-sdl was ˜4 fold less. This is possiblydue to higher S. dysenteriae LPS expression in Ty21a-sdl compared toTy21a-sd. However, these results confirm the stable expression of bothheterologous S. dysenteriae O-antigen and homologous Ty21a LPS inTy21a-sd and Ty21a-sdl.

Mouse Protection from Virulent S. dysenteriae Challenge

Immunized and control mice were challenged intraperitoneally, 2 weeksafter final immunization, with 1.5×10⁶ CFU/ml of freshly grown,mid-log-phase virulent S. dysenteriae 1 strain 1617ΔstxA in 0.5 ml(7.5×10⁵ CFU per mouse) of 5% hog gastric mucin (Sigma) dissolved insterile saline (i.e., approximately 10 times the 50% lethal infectiousdose [LD50]). Survival was monitored for 72 h (FIG. 8). Although thischallenge resulted in 100% mortality in saline-immunized mice, only 50%mortality resulted in Ty21a-alone immunized mice. This may be due to lowlevel of S. dysenteriae LPS cross-reacting antibodies elicited by Ty21a,as determined by ELISA, providing some protection. Also, a low level ofprotection conferred by Ty21 has been observed before (Noriega, et al.1999 Infection and immunity 67:782-788). The mice immunized with therecombineered vaccine strain Ty21a-Sd and Ty21a-Sdl were 100% and 70%,respectively, protected against the S. dysenteriae challenge.

Example 9. Chromosomal Integration of Shigella flexneri 2a and 3aO-Antigen Genes

There are 14 S. flexneri serotypes on the basis of antigenicdeterminants on the O-antigen. In all of the S. flexneri serotypes, withthe exception of serotype 6, all share a common polysaccharide backbonecomprising repeating units of the tetrasaccharideN-acetylglucosamine-rhamnose-rhamnose-rhamnose (FIG. 9). The basicO-antigen backbone is called the serotype Y. The genes involved in thebiosynthesis of the O-antigen backbone are located in the rfb operon(˜10 kb) flanked by gnd and galF genes. Modification of the O-antigenbackbone by the addition of glucosyl and/or O-acetyl groups to differentsugars in the tetrasaccharide gives rise to different serotypes. Thegenes involved in the O-antigen modification are encoded on temperatebacteriophages (Allison, et al. 2000 Trends in Microbiol 8:17-23). Twogenes encoded on lysogenic bacteriophage SfII were found to be essentialfor 2a serotype conversion. These genes are bgt, which encodes aputative bactoprenol glucosyl transferase, and gtrII, encoding theputative type II antigen determining glucosyl transferase (Mavris, etal. 1997 Mol Microbiol 26:939-950).

S. flexneri 3b O-antigen contains an O-acetyl modification, and S.flexneri 3a O-antigen contains a glucosyl modification in addition tothe O-acetyl modification. The gene, oac, encoded on lysogenicbacteriophage Sf6, was found to be essential for the 3b serotypeconversion from the Y serotype (Clark, et al. 1991 Gene 107:43-52). Theglucosylation gene cluster (gtrA, gtrB, and gtrX) encoded bybacteriophage Sfx is involved in O-antigen modification of serotype Y toX and serotype 3b to 3a. The first gene of the glucosylation genecluster gtrA encodes a small highly hydrophobic protein involved in thetranslocation of lipid-linked glucose across the cytoplasmic membrane.The gene gtrB encodes an enzyme catalyzing the transfer of the glucoseresidue from UDP-glucose to a lipid carrier, and gtrX encodes abacteriophage-specific glucosyltransferase for the final step ofattaching the glucosyl molecules onto the correct sugar residue of theO-antigen repeating unit (Guan, et al. 1999 Mol Microbiol 26:939-950).

Cloning S. flexneri 2a and 3a O-Antigen Genes into pMD-TV

Since cloning ˜10 kb rfb operon was challenging, ˜6 kb of the rfb operonwas cloned into low copy plasmid pMD-TV (Dharmasena, et al. 2013 Intl JMed Microbiol 303:105-113), using KpnI and XhoI, followed by remaining 4kB of the rfb operon using XhoI and BamHI. The S. flexneri 4 kb and 6 kbO-antigen gene regions were PCR amplified from S. flexneri 2a 2457genomic DNA using prMD3-2a.rfb.F-prMD16.R and prMD18.rfb.5021.FprMD4-2a.rfb.R primer pairs (Table F, below), respectively.

TABLE F PRMD3- GATCGGGATCCTAATGAAAATCTGACCGGATGTAACG 2A.RFB.FGTTG (SEQ. ID NO: 34) PRMD16.504 GATCACTCGAGCGAGAAATCCTAGCG 2.R(SEQ. ID NO: 35) PRMD18.RF CCCCTCGCTAGGATTTCTCGCTCGAG B.F(SEQ. ID NO: 36) PRMD4- GATCGGGTACCTTGTTTTCTGAGCGAATATATATAAG 2A.RFB.R(SEQ. ID NO: 37) PRMD1- GATCGGCGGCCGCGACCCAAAATGGACTATGACAGAA 2AGTR-FAGATCTGATATTTTTCCTCGCAAAAATGAAAATATCT CTTGTCGTTC (SEQ. ID NO: 38) PRMD2-GATCGGGATCCTAAATATTAAATGGAAGCC 2AGTR-R (SEQ. ID NO: 39) PRMD47.3A. ATAAGAATGCGGCCGCACTGGCTGGACCCAAAATGG OAC.NOTI (SEQ. ID NO: 40)PRMD30.3A.  GATCAGGATCCTCAATCCAGGGATAATTTAGGCGAAC BAMHI.R(SEQ. ID NO: 41) PRMD120.SF  GATCAGGATCCATCGATTGAGACTTGGATGATAGACTX.BAMHI.F TCATG (SEQ. ID NO: 42) PRMD121..S GATCAGGATCCTTATTTTTTTATTAAATCAAGAGTTA FX.BAMH.RACCATGGAGGGAG (SEQ. ID NO: 43) PRMD87.VETTAGAAAGAATTAGTGCCGCGGGTCAAAAAGC XA.R.32BP (SEQ. ID NO: 44) PRMD133.TCCGTTCCTCATTGATTTGATTGCTAAC VID.F (SEQ. ID NO: 45) PRMD123.Y.R CGGCAACATAAGTAATTTGCTCACG (SEQ. ID NO: 46) PRMD124.F.CACGCAATATTTCAATGATGGCAAC TVID (SEQ. ID NO: 47) PRMD92.VEGTTGCGGTAATGGTATAACGAAATAACAGATAC XB.R.17800  (SEQ. ID NO: 48)PRMD118.R. TATTTATTATGTGACGAACAACAGCAGAACC 2AY (SEQ. ID NO: 49)PRMD152.2A  AATTGACTCTGTGGCATCTTTACTTCCGTCATTTATG .LPP.RAATACAATTTCTACTTCATATGGCTTCAACTCTTGGAATTCACGTACCGTTTTATAGAAAACAGGTATCGCTTCTTCTTCATTGAAGACAGGAACGACAAGAGATATTTTCATATGTCCTCTCCTTTCATTATTAATACCCTCTAGAG TTC (SEQ. ID NO: 50)

The resulting PCR 6 kb product and pMD-TV plasmid were digested withKpnI and XhoI, PCR-purified, and ligated to construct pMD-TV.2a.6,followed by digestion of 4 Kb PCR product and pMD-TV.2a.6 with XhoI andBamHI to construct pMD-TV-Y.

In order to express S. flexneri 2a O-antigen, the bacteriophageSfII-encoded bgt-gtrII were cloned into pMD-TV-Y upstream of the S.flexneri 2a rfb region. First bgt-gtrII was PCR-amplified from S.flexneri 2a 2457 genomic DNA with primers that contained NotI and BamHI(prMD1-2agtr-F and prMD2-2agtr-R, FIG. 9). The resulting PCR product andpMD-TV-Y were digested with NotI and BamHI, PCR-purified, and ligated toconstruct pMD-TV-2a.

In order to express S. flexneri 3a O-antigen, the S. flexneri 2a rfbregion, bacteriophage Sf6 encoded oac and bacteriophage SfX-encodedgtrX, gtrA and gtrB were cloned, with their cognate promoters, tandemlyinto the pMD-TV vector. The SF6-encoded oac was PCR-amplified from S.flexneri 3a J99 genomic DNA with primers that contained NotI and BamHIsites (prMD47.3a.OAC.NotI and prMD30.3a.BamHI.R, FIG. 9). The resultingPCR product and pMD-TV-Y were digested with NotI and BamHI,PCR-purified, and ligated to construct pMD-TV-3b. Similarly, gtrX, gtrA,and gtrB gene cluster was PCR-amplified from S. flexneri 3a J99 genomicDNA with primers that contained BamHI site (prMD120.sfx.bamHI.F andprMD121.sfx.bamH.R, FIG. 9) and cloned into the BamHI site of pMD-TV-3bto construct pMD-TV-3a. All of the constructs were confirmed by PCR andsequencing.

Expression of S. flexneri 2a and 3a O-Antigen from the Plasmid

Expression of S. flexneri 2a and 3a O-antigen from pMD-TV-2a andpMD-TV-3a, respectively, in E. coli and Ty21a, was confirmed by slideagglutination and by Western blotting. pMD-TV-2a in E. coli or Ty21areacts with S. flexneri type II specific anti-sera (FIG. 10) and alsoagglutinated with S. flexneri 3,4 epitope specific anti-sera. Thus,pMD-TV-2a contains all of the genes required for stable S. flexneri 2aO-antigen expression (Dharmasena, et al. 2012 Am Soc Microbiol mtg, SanFrancisco, Calif.).

pMD-TV-3a in E. coli or Ty21a reacts with S. flexneri type III specificanti-sera (FIG. 10). pMD-TV-3a strains also agglutinated with S.flexneri 6 and 7, 8 epitope specific anti-sera. Thus, pMD-TV-3a containsall of the genes required for stable S. flexneri 3a O-antigenexpression.

Integrating S. flexneri O-Antigen Genes into Ty21a Chromosome

The S. flexneri O-antigen Y serotype backbone genes that were clonedinto pMD-TV (pMD-TV-Y) were used as template for PCR with tviD forward(prMD133.tviD.F) primer and vexA reverse (prMD87.vexA.R.32 bp) primer.The PCR-amplified region that contains part of tviD gene, Kan cassetteflanked by FTR sites, S. flexneri rfb genes and vexA gene was 13,653 bp.These PCR products were transformed into Ty21a competent cellsexpressing 2 red proteins from pKD46 and selected for Kan resistance asdescribed in (Dharmasena, et al. 2013). PCR with primers upstream anddownstream of the site of integration prMD92 and prMD124 resulted in a˜14.5 Kb band. After removing the Kanr cassette as described in(Dharmasena, et al. 2013), antibiotic-sensitive chromosomal integrantswere further analyzed by PCR for deletion of the Kan cassette andsequenced the PCR product (strain MD 114/Ty21a-Y˜13 Kb band) obtainedfrom primers prMD92 and prMD124. The sequence of this PCR was comparedto S. flexneri 2a 2457 (GenBank accession no. AE014073.1, completegenome 4,599,354 bp, reference: Wei, et al. 2003 Infect Immun71(5):2775-2786), and only a single A insertion was detected in theintergenic region upstream of gene rfbC (FIG. 11), which did not affectthe O-antigen expression determined by Western blot with anti-S.flexneri 1-6 antibody.

In order to integrate S. flexneri 2a modifying enzymes bgt and gtrIIupstream of rfb operon in Ty21a-Y, pMD-TV-2a was used as template forPCR with tviD forward primer (prMD133.tviD.F) and rfb operon reverseprimer (prMD118.R.2aY). The PCR-amplified region that contains part oftviD gene, Kan cassette flanked by FTR sites, S. flexneri 2a modifyingenzymes bgt and gtrII, and first ˜500 bp of the rfb operon was 5,132 bp.These PCR products were transformed into Ty21a-Y competent cellsexpressing 2 red proteins and selected for Kan resistance as before. PCRwith primers upstream and downstream of the site of integration prMD92and prMD124 resulted in a ˜17 Kb band. After removing the Kan^(r)cassette, antibiotic-sensitive chromosomal integrants were furtheranalyzed by PCR for deletion of the Kan cassette and sequenced the PCRproduct (strain MD 194/Ty21a-2a ˜15.5 Kb band) obtained from primersprMD92 and prMD124. The sequence of this PCR was compared to SFII codedbgt-gtrII region and rfb region of S. flexneri 2a 2457, and noadditional mutations were found. Also, S. flexneri 2a expression wasdetermined by Western blot with S. flexneri type II specific anti-serashowed only weak expression compared to the plasmid expression (FIG.12). This may be due to multiple copies of the plasmid (˜5 per cell)expressing more S. flexneri 2a O-antigen compared to only one copy of S.flexneri 2a O-antigen biosynthetic genes integrated into the Ty21achromosome in Ty21-2a strain (FIG. 12).

Similarly, S. flexneri 3a modifying enzymes gtrX, gtrA, and gtrB genecluster and oac were integrated upstream of the rfb operon in theTy21a-Y strain as described before using pMD-TV-3a as template for PCR.The PCR product that was transformed into Ty21a-Y competent cellsexpressing λ red was 6448 bp. After integration, PCR with primersupstream and downstream of the site of integration prMD92 and prMD124resulted in a ˜18.5 Kb band and after removing the Kan^(r)cassette(strain MD 194/Ty21a-3a) resulted in a ˜17 Kb band. The sequence of thisPCR was compared to bacteriophage Sf6-encoded oac and bacteriophageSfX-encoded gtrX, gtrA, and gtrB and S. flexneri 2a 2457, and noadditional mutations were found. Also, S. flexneri 3a expression wasdetermined by Western blot with S. flexneri type III specific anti-serashowed only weak expression compared to the plasmid expression as S.flexneri 2a (FIG. 12).

Replacing Bgt Native Promoter with lpp

Attempts were made to increase the S. flexneri 2a LPS expression byreplacing the native bgt promoter with the lpp promoter, which is ahighly transcribed constitutively active promoter. Ty21a-sdl (S.dysenteriae O-antigen biosynthesis genes expressed from lpp promoter)was used as template for PCR with tviD forward primer and lpp promoterreverse primer with 150 bp bgt homology extension (Table F). ThePCR-amplified region contains part of tviD gene, Kan cassette flanked byFTR sites, 200 bp lpp promoter, and first 150 bp bgt gene. The PCRproducts were transformed into Ty21a-2a (MD194) competent cellsexpressing λ red proteins and selected for Kan resistance, removed theKan cassette, and sequenced as described before to construct Ty21a-2al(MD212). Western blot with S. flexneri type II specific anti-sera showsthat Ty21a-2al (MD212) expresses higher S. flexneri 2a LPS than that ofTy21a-2a (MD194), but still less than plasmid expression (Ty21apMD-TV-2a) (FIG. 13).

Additional efforts are being made to carry out animal experimentsemploying new constructs.

REFERENCES

-   1. Kotloff, K. L., Winickoff, J. P., Ivanoff, B., Clemens, J. D.,    Swerdlow, D. L., Sansonetti, P. J., Adak, G. K., and    Levine, M. M. (1999) Bulletin of the World Health Organization 77,    651-666-   2. Kweon, M. N. (2008) Current opinion in infectious diseases 21,    313-318-   3. Mead, P. S., Slutsker, L., Dietz, V., McCaig, L. F., Bresee, J.    S., Shapiro, C., Griffin, P. M., and Tauxe, R. V. (1999) Emerging    infectious diseases 5, 607-625-   4. Putthasri, W., Lertiendumrong, J., Chompook, P.,    Tangcharoensathien, V., and Coker, R. (2009) Emerging infectious    diseases 15, 423-432-   5. WHO. (2005). in Guidelines for the control of shigellosis,    including epidemics due to Shigella dysenteriae type 1, World Health    Organization, Geneva, Switzerland-   6. Kaminski, R. W., and Oaks, E. V. (2009) Expert review of vaccines    8, 1693-1704-   7. Ferreccio, C., Prado, V., Ojeda, A., Cayyazo, M., Abrego, P.,    Guers, L., and Levine, M. M. (1991) American journal of epidemiology    134, 614-627-   8. DuPont, H. L., Hornick, R. B., Snyder, M. J., Libonati, J. P.,    Formal, S. B., and Gangarosa, E. J. (1972) The Journal of infectious    diseases 125, 5-11-   9. DuPont, H. L., Hornick, R. B., Snyder, M. J., Libonati, J. P.,    Formal, S. B., and Gangarosa, E. J. (1972) The Journal of infectious    diseases 125, 12-16-   10. Noriega, F. R., Liao, F. M., Maneval, D. R., Ren, S., Formal, S.    B., and Levine, M. M. (1999) Infection and immunity 67, 782-788-   11. Xu de, Q., Cisar, J. O., Osorio, M., Wai, T. T., and    Kopecko, D. J. (2007) Vaccine 25, 6167-6175-   12. Xu, D. Q., Cisar, J. O., Ambulos Jr, N., Jr., Burr, D. H., and    Kopecko, D. J. (2002) Infection and immunity 70, 4414-4423-   13. Osorio, M., Wu, Y., Singh, S., Merkel, T. J., Bhattacharyya, S.,    Blake, M. S., and Kopecko, D. J. (2009) Infection and immunity 77,    1475-1482-   14. Kopecko, D. J., Sieber, H., Ures, J. A., Furer, A., Schlup, J.,    Knof, U., Collioud, A., Xu, D., Colburn, K., and Dietrich, G. (2009)    International journal of medical microbiology: IJMM 299, 233-246-   15. D'Amelio, R., Tagliabue, A., Nencioni, L., Di Addario, A.,    Villa, L., Manganaro, M., Boraschi, D., Le Moli, S., Nisini, R., and    Matricardi, P. M. (1988) Infection and immunity 56, 2731-2735-   16. Seid, R. C., Jr., Kopecko, D. J., Sadoff, J. C., Schneider, H.,    Baron, L. S., and Formal, S. B. (1984) The Journal of biological    chemistry 259, 9028-9034-   17. Formal, S. B., Baron, L. S., Kopecko, D. J., Washington, O.,    Powell, C., and Life, C. A. (1981) Infection and immunity 34,    746-750-   18. Black, R. E., Levine, M. M., Clements, M. L., Losonsky, G.,    Herrington, D., Berman, S., and Formal, S. B. (1987) The Journal of    infectious diseases 155, 1260-1265-   19. Herrington, D. A., Van de Verg, L., Formal, S. B., Hale, T. L.,    Tall, B. D., Cryz, S. J., Tramont, E. C., and Levine, M. M. (1990)    Vaccine 8, 353-357-   20. Van de Verg, L., Herrington, D. A., Murphy, J. R., Wasserman, S.    S., Formal, S. B., and Levine, M. M. (1990) Infection and immunity    58, 2002-2004-   21. Datsenko, K. A., and Wanner, B. L. (2000) Proceedings of the    National Academy of Sciences of the United States of America 97,    6640-6645-   22. Cherepanov, P. P., and Wackernagel, W. (1995) Gene 158, 9-14-   23. Murphy, K. C. (1998) Journal of bacteriology 180, 2063-2071-   24. Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M.,    Datta, S., and Court, D. L. (2007) Methods in enzymology 421,    171-199-   25. Uzzau, S., Figueroa-Bossi, N., Rubino, S., and Bossi, L. (2001)    Proceedings of the National Academy of Sciences of the United States    of America 98, 15264-15269-   26. Yu, B., Yang, M., Wong, H. Y., Watt, R. M., Song, E., Zheng, B.    J., Yuen, K. Y., and Huang, J. D. (2011) Applied microbiology and    biotechnology 91, 177-188-   27. Ohtake, S., Martin, R., Saxena, A., Pham, B., Chiueh, G.,    Osorio, M., Kopecko, D., Xu, D., Lechuga-Ballesteros, D., and    Truong-Le, V. (2011) Vaccine 29, 2761-2771-   28. Germanier, R., and Fuer, E. (1975) The Journal of infectious    diseases 131, 553-558-   29. Kopecko, D. J., Washington, O., and Formal, S. B. (1980)    Infection and immunity 29, 207-214-   30. Churchward, G., Belin, D., and Nagamine, Y. (1984) Gene 31,    165-171

The invention claimed is:
 1. A Salmonella Typhi Ty21a comprising aShigella sonnei form 1 O-antigen biosynthetic gene region inserted intothe Salmonella Typhi Ty21a chromosome, wherein: a) heterologous Shigellasonnei form 1 O-antigen is stably expressed together with or withouthomologous Salmonella Typhi O-antigen; b) immune protection is elicitedagainst virulent Shigella sonnei challenge; and c) immune protection iselicited against virulent Salmonella Typhi challenge when heterologousShigella sonnei form 1 O-antigen is stably expressed together withhomologous Salmonella Typhi O-antigen; and d) the region comprises a DNAsequence, wherein the DNA sequence comprises: 1) the DNA sequence as setout in SEQ ID NO:2, or 2) a DNA sequence that shares at least about 90%sequence identity with the DNA sequence set out in SEQ ID NO:2.
 2. TheSalmonella Typhi Ty21a of claim 1, further comprising an O-antigenbiosynthetic gene region from a bacterial strain selected from the groupconsisting of: Shigella species, Escherichia coli serotypes, Salmonellaenterica serovars, Vibrio cholerae serotypes, Enterobacter species,Yersinia species, Plesiomonas species, and Pseudomonas species.
 3. ASalmonella Typhi Ty21a comprising a Shigella dysenteriae 1 O-antigenbiosynthetic gene region inserted into the Salmonella Typhi Ty21achromosome, wherein: a) heterologous Shigella dysenteriae serotype 1O-antigen is stably expressed together with or without homologousSalmonella Typhi O-antigen; b) immune protection is elicited againstvirulent Shigella dysenteriae challenge; and c) immune protection iselicited against virulent Salmonella Typhi challenge when heterologousShigella dysenteriae serotype 1 O-antigen is stably expressed togetherwith homologous Salmonella Typhi O-antigen; and d) the region comprisesa DNA sequence, wherein the DNA sequence comprises: 1) the DNA sequenceas set out in SEQ ID NO:33, or 2) a DNA sequence that shares at leastabout 90% sequence identity with the DNA sequence set out in SEQ IDNO:33.
 4. The Salmonella Typhi Ty21a of claim 3, further comprising anO-antigen biosynthetic gene region from a bacterial strain selected fromthe group consisting of: Shigella species, Escherichia coli serotypes,Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacterspecies, Yersinia species, Plesiomonas species, and Pseudomonas species.5. A plasmid construct having i) a DNA sequence as set out in SEQ ID NO:1 or ii) a DNA sequence that shares at least about 90% sequence identitywith the DNA sequence set out in SEQ ID NO:1.
 6. The plasmid constructof claim 5, further comprising a Shigella sonnei O-antigen biosyntheticgene region or a Shigella dysenteriae 1 O-antigen biosynthetic generegion.
 7. A method of recombineering a large antigenic gene region intoa bacterial chromosome, comprising: i) cloning the region into a vectorcontaining: ia) a genetically selectable marker flanked 5′ and 3′ by anFRT site, respectively; ib) a multiple cloning site downstream of the 3′FRT site; and ic) two sites of chromosome homology, one of the twolocated upstream of the 5′ FRT site, and one of the two locateddownstream of the multiple cloning site; ii) integrating the region intothe bacterial chromosome using X, red recombination; iii) selecting forthe genetically selectable marker; and iv) removing the selectablemarker, thus recombineering the large antigenic gene region into thechromosome.
 8. The method of claim 7, wherein the large antigenic generegion is about 5 to about 20 kb long.
 9. The method of claim 7, whereinthe vector is selected from the group consisting of a plasmid, phage,phasmid, and cosmid construct.
 10. The method of claim 9, wherein theplasmid construct has i) a DNA sequence as set out in SEQ ID NO: 1 orii) a DNA sequence that shares at least about 90% sequence identity withthe DNA sequence set out in SEQ ID NO:1.
 11. The method of claim 7,wherein the bacterial chromosome is from Salmonella Typhi Ty21a.
 12. Themethod of claim 7, wherein the genetically selectable marker is anantibiotic resistance marker.
 13. The method of claim 12, wherein theantibiotic resistance marker is kanamycin.
 14. The method of claim 7,wherein the large antigenic gene region is selected from the groupconsisting of a Shigella sonnei O-antigen biosynthetic gene region, aShigella dysenteriae 1 O-antigen biosynthetic gene region, a Shigellaflexneri 2a 0-antigen biosynthetic gene region, and a Shigella flexneri3a O-antigen biosynthetic gene region.
 15. The method of claim 7,wherein the large antigenic gene region is engineered between the twosites of chromosome homology, each site comprising between about 500 toabout 1000 bp regions of bacterial chromosome homology before step ii.16. The method of claim 13, wherein the kanamycin resistance gene isremoved via recombination induced following transformation ofchromosomal integrants of step ii with pCP20.
 17. A vaccine comprising apharmaceutically acceptable solid carrier and Salmonella Typhi Ty21acomprising a Shigella flexneri 2a O-antigen biosynthetic gene regioninserted into the Salmonella Typhi Ty21a chromosome, wherein: a)heterologous Shigella flexneri 2a O-antigen is stably expressed togetherwith or without homologous Salmonella Typhi O-antigen; b) immuneprotection is elicited against virulent Shigella flexneri 2a challenge;and c) immune protection is elicited against virulent Salmonella Typhichallenge when heterologous Shigella flexneri 2a O-antigen is stablyexpressed together with homologous Salmonella Typhi O-antigen.
 18. Avaccine comprising a pharmaceutically acceptable solid carrier andSalmonella Typhi Ty21a comprising a Shigella flexneri 3a O-antigenbiosynthetic gene region inserted into the Salmonella Typhi Ty21achromosome, wherein: a) heterologous Shigella flexneri 3a O-antigen isstably expressed together with or without homologous Salmonella TyphiO-antigen; b) immune protection is elicited against virulent Shigellaflexneri 3a challenge; and c) immune protection is elicited againstvirulent Salmonella Typhi challenge when heterologous Shigella flexneri3a O-antigen is stably expressed together with homologous SalmonellaTyphi O-antigen.
 19. A vaccine comprising the Salmonella Typhi Ty21a ofclaim 1 in combination with a physiologically acceptable carrier.
 20. Amethod of treating at least one bacterial infection comprisingadministering a prophylactically or therapeutically effective amount ofthe Salmonella Typhi Ty21a of claim 1 to a subject, thus treating the atleast one bacterial infection.
 21. The Salmonella Typhi Ty21a of claim1, wherein the Shigella sonnei form 1 O-antigen biosynthetic gene regionis partially or wholly chemically synthesized.
 22. The plasmid constructof claim 5, wherein the DNA sequence is partially or wholly chemicallysynthesized.